  Ice and cooling mixtures

Contributor:

Zappe, H

Reference:

-

Ice

Always make an ice/water slurry for temporary enzyme storage (or use one of the Stratagene freezer boxes).  The mixture will have a temperature of 0 - 1°C, whereas ice chips, with all the trapped air, has a much poorer heat/cold transfer and solutions remain at about 4°C or more.

Cooling mixtures

Taken from the Nalge Company, Division of Sybron Corporation, Rochester, New York 14606, USA, makers of Dewar flasks for liquid N2.  The actual temperature is affected by impurities and is therefore approximate.

Mixture	°C	°F

Air directly above ice	15	39

Ice-water slurry	0	32

NaCl-Ice (33 g NaCl / 100 g ice	-21	-6

CaCl2-ice (100 g CaCl2 / 100 g ice)	-40	-40

Chloroform / liquid N2 (slush)	-64	-83

Dry ice / acetone (a)	-78	-108

Dry ice / isopropanol (a)	-78	-108

Toluene / liquid N2 (slush)	-95	-139

Pentane liquid N2 (slush)	-130	-202

N2 vapour above liquid N2	-140	-220

Isopentane / liquid N2	-160	-256

Liquid N2	-196	-320

(a) - acetone is more volatile than isopropanol and may attack the container or container lid being used (e.g. polystyrene), while isopropanol will not.

Use Dewar flasks for any mixtures containing dry ice or liquid N2.


  Nucleic acid weight data

Contributor:

Zappe, H

Reference:

-

Average weight of a DNA base-pair (sodium salt = 660 daltons)

Concentrations of DNA and RNA solutions

1.0 A260 unit dsDNA = 50 μg/ml

1.0 A260 unit ssDNA = 33 μg/ml

1.0 A260 unit ssRNA = 40 μg/ml

Double-stranded DNA

1 pmole = (0.662 X length in kb) μg

e.g. for 1 kb DNA

1 pmole = (0.662 X 1) = 0.662 μg

1 pmole  pUC18 DNA = 1.78 μg (pUC 18 is 2.686 kb)

Primers and oligonucleotides

1 pmole = (0.331 X length in bp) ng

e.g. for a 20 mer

1 pmole = (0.331 X 20) ng = 6.62 ng


Lambda DNA standards, diagrams
  λ DNA size standards

Contributor:

Babb, B

Reference:

-

Sizes of λ phage DNA cut with variou restriction enzymes to produce a ladder of known size standards.  The following pictures show what the digests look like for various commonly used enzymes, with the sizes given in kb.


  Genetic code

Contributor:

Babb, B

Reference:

many

U	C	A	G

U	uuu	phe	ucu	ser	uau	tyr	ugu	cys	u

uuc	phe	ucc	ser	uac	tyr	ugc	cys	c

uua	leu	uca	ser	uaa	stop	uga	stop	a

uug	leu	ucg	ser	uag	stop	ugg	trp	g

C	cuu	leu	ccu	pro	cau	his	cgu	arg	u

cuc	leu	ccc	pro	cac	his	cgc	arg	c

cua	leu	cca	pro	caa	gln	cga	arg	a

cug	leu	ccg	pro	cag	gln	cgg	arg	g

A	auu	ile	acu	thr	aau	asn	agu	ser	u

auc	ile	acc	thr	aac	asn	agc	ser	c

aua	ile	aca	thr	aaa	lys	aga	arg	a

aug	met	acg	thr	aag	lys	agg	arg	g

G	guu	val	gcu	ala	gau	asp	ggu	gly	u

guc	val	gcc	ala	gac	asp	ggc	gly	c

gua	val	gca	ala	gaa	glu	gga	gly	a

gug	val	gcg	ala	gag	glu	ggg	gly	g


Notes on radioactivity
  Radioactivity

Contributor:

Babb, B

Reference:

-

Caution:

 consult your local radiation officer!

Isotope	particle emitted	Half life

14C	β	5730yrs

3H	β	12.3yrs

125I	γ	60d

32P	β	14.3d

35S	β	87.4d

1 Becquerel = 1 disintegration/sec

1 μCi = 3.70x10**4 Becquerels

1 Becquerel = 2.70 x10**-5 μCi


  Restriction enzyme sites

Contributor:

Babb, B

Reference:

GCG package, University of Wisconsin

Overhang codes
5'overhang 	> 0
blunt      	= 0
3'overhang 	< 0
unknown    	= ?

Restriction enzyme sites and overhangs

Enzyme	Site	Overhang

A

AccI	GT'mk_AC	2

AhaII	Gr'CG_yC	2

AluI	AG'CT	0

AlwI	GGATCnnnn'n_	1

ApaI	G_GGCC'C	-4

ApaLI	G'TGCA_C	4

AsuI	G'GnC_C	3

AsuII	TT'CG_AA	2

AvaI	C'yCGr_G	4

B

BalI	TGG'CCA	0

BamHI	G'GATC_C	4

BclI	T'GATC_A	4

BglI	GCCn_nnn'nGGC	-3

BglII	A'GATC_T	4

BstI	G'GATC_C	4

BstEII	G'GTnAC_C	5

BstXI	CCAn_nnnn'nTGG	-4

C

CfoI	G_CG'C	-2

ClaI	AT'CG_AT	2

D

DraI	TTT'AAA	0

E

EcoRI	G'AATT_C	4

EcoRV	GAT'ATC	0

F

FokI	GGATGnnnnnnnnn'nnnn_	4

G

H

HaeIII	GG'CC	0

HhaI	G_CG'C	-2

HincII	GTy'rAC	0

HindII	GTy'rAC	0

HindIII	A'AGCT_T	4

HinfI	G'AnT_C	3

HinfIII	CGAAT	?

HpaI	GTT'AAC	0

HpaII	C'CG_G	2

I

J

K

KpnI	G_GTAC'C	-4

L

M

MboI	'GATC_	4

MboII	GAAGAnnnnnnn_n'	-1

MluI	A'CGCG_T	4

MraI	CCGCGG	?

N

NaeI	GCC'GGC	0

NcoI	C'CATG_G	4

NdeI	CA'TA_TG	2

NotI	GC'GGCC_GC	4

NruI	TCG'CGA	0

O

P

PaeR7I	C'TCGA_G	4

PstI	C_TGCA'G	-4

PvuI	CG_AT'CG	-2

PvuII	CAG'CTG	0

Q

R

RsaI	GT'AC	0

S

SacI	G_AGCT'C	-4

SalI	G'TCGA_C	4

Sau96I	G'GnC_C	3

Sau3AI	'GATC_	4

ScaI	AGT'ACT	0

SfiI	GGCCn_nnn'nGGCC	-3

SmaI	CCC'GGG	0

SpeI	A'CTAG_T	4

SphI	G_CATG'C	-4

SspI	AAT'ATT	0

SstI	G_AGCT'C	-4

SstII	CC_GC'GG	-2

StuI	AGG'CCT	0

StyI	C'CwwG_G	4

T

TaqI	T'CG_A	2

U

V

W

X

XbaI	T'CTAG_A	4

XhoI	C'TCGA_G	4

XmaI	C'CCGG_G	4

XmnI	GAAnn'nnTTC	0

Y

Z


Amino acid codes, 3 and 1 letter
  Amino acid codes

Contributor:

Babb, B

Reference:

Sambrook et al. (199?)

Amino acid	Codes

Alanine	Ala	A

Arginine	Arg	R

Asparagine	Asn	N

Aspartic acid	Asp	D

Cysteine	Cys	C

Glutamine	Gln	Q

Glutamic acid	Glu	E

Glycine	Gly	G

Histidine	His	H

Isoleucine	Ile	I

Leucine	Leu	L

Lysine	Lys	K

Methionine	Met	M

Phenylalanine	Phe	F

Proline	Pro	P

Serine	Ser	S

Threonine	Thr	T

Tryptophan	Trp	W

Tyrosine	Tyr	Y

Valine	Val	V


   Appendix

Partial fill-in sites and restriction enzymes

1.	description

2.	restriction enzyme

3.	fill-in site  of 2bp

4.	fill-in site  of 1bp


Partial fill-in, overhang sequence listing
  Partial fill-in

Contributor:

Babb, B

Reference:

New England Biolabs catalog

By filling in with klenow and not all nucleotides (see complete Fill in), one can generate DNA fragments in which the parentals will not be able to undergo circle closure.  This effectively forces the vector to take up the insert molecule.  However only certain enzymes can be used - namely those that generate 5' overhangs.

List of restriction enzymes by
restriction enzyme
fill-in site of 2bp
fill-in site of 1bp


Partial fill-in, overhang sequence listing by 2bp overhang sequence
  Partial fill-in

Order by fill-in site (2bp)

Contributor:

Babb, B

Reference:

New England Biolabs catalog

For example ApaLI on partial fillin can generate a 5'-TG-3' overhang, which will anneal to a 3'AC5' overhang, which the table shows can be generated by NcoI

N N A A	T T N     N	AflII
N N A C	T G N     N	ApaLI
N N A G	T C N     N	XhoI  SalI  PaeR71
N N A T	T A N     N	NdeI  AseI  MseI
N N C A	G T N     N	BsiW1  BsrGI
N N C C	G G N     N	NotI  EaeI
N N C G	G C N     N
N N C T	G A N     N	Sau3AI  MboI  BstI  BglII  BclI  BamHI
N N G A	C T N     N	XbaI  SpeI  AvrII  NheI
N N G C	C G N     N	HpaII  ClaI  AsuII  AhaII  TaqI  BsqHI
N N G G	C C N     N	XmnI  AgeI  BsaWI  BspEI  NgoMI
N N G T	C A N     N	NcoI  BspHI  EagI
N N T A	A T N     N
N N T C	A G N     N	HindIII
N N T G	A C N     N
N N T T	A A N     N	EcoRI  ApoI


Partial fill-in, overhang sequence listing by restriction enzyme
  Partial fill-in

Order by restriction enzyme

Contributor:

Babb, B

Reference:

New England Biolabs catalog

fill-in sites are listed 5' to 3' eg ApaLI after fill in will look like such:

NNNN NNNNGT	or	TGNNNN   NNNN
enzyme and overhang	double	single

ApaLI	TGN	TN

AseI 	TAN	TN

AflII	TTN	-N

AgeI	CCN	-N

AhaII	CGN	CN

ApoI	AAN	-N

AsuII	CGN	CN

AvaII	GN

AvrII	CTN	CN

BamHI	GAN	GN

BclI	GAN	GN

BfaI 	-	TN

BglII	GAN	GN

BsaHI	CGN	CN

BsiWI	GTN	GN

BsaWI	CCN	-N

BsrGI	GTN	GN

BspEI	CCN	-N

BspHI	CAN	CN

BstI	GAN	GN

ClaI	CGN	CN

EaeI	GGN	-N

EagI	CAN	CN

EcoRI	AAN	-N

HindIII	AGN	AN

HpaII	CGN	CN

MboI	GAN	GN

MseI	TAN	TN

MspI	-	CN

NarI	-N	CN

NcoI	CAN	CN

NdeI	TAN	TN

NgoMI	CCN	-N

NheI	CTN	CN

NotI	GGN	-N

NN

PaeR71 	TCN	TN

SalI	TCN	TN

Sau3A1	GAN	GN

SpeI	CTN	CN

TaqI	CGN	CN

XbaI	CTN	CN

XhoI	TCN	TN

XmaI	CCN	-N


Partial fill-in, overhang sequence listing by 1bp overhang sequence
  Partial fill-in

Order by fill-in site (1bp)

Contributor:

Babb, B

Reference:

New England Biolabs catalog

ApaLi generates a T overhang, which will anneal to a A overhang, such as generated by HindIII

N N A	T N   N	ApaLI  AseI  BfaI  MseI NdeI PaeR71  SalI
N N C	G N   N	AvaII  BamHI  BclI  BglII  BsiWI  MboI  Sau3A1
N N G	C N   N	AvrII  BsaHI  BspHI  HpaII  MspI  NarI  NcoI  NheI  SpeI  XbaI
N N T	A N   N	HindIII


DNA shortening with Bal31 nuclease, description
  Bal31 shortening

Protocol       Related

Description;

Bal31 is a exonuclease that chomps in both directions,  The idea is to shorten your insert and vector, then cut out the shortened fragment and clone it.

Requirements

The insert in pUC19 or SK (KS) should be bracketed by 2 unique sites (e.g. EcoRI and HindIII).  Sets of deletions from both sides are generated by separate digestion of EcoRI and HindIII cut plasmid with BAL-31 and then cutting with HindIII and EcoRI respectively, to release the shortened fragments from the remainder of the vector, and subcloning to fresh vector cleaved with HindIII or EcoRI and SmaI (BAL-31 leaves blunt ends on a high percentage of fragments).

Shortening of DNA using ExoIII nuclease.  Insert to be shortened must have two correct types of r/e sites on side of insert to be shortened.  One with a ...


DNA shortening with Bal31 nuclease, protocol
  Bal31 shortening

Description        Related

Contributor:

Zappe, H

Reference:

-

1.	Digest 30 μg of pUC19 plus insert in a final volume of 200 μl.  Check by electrophoresis for complete digestion.

2.	Prepare 10 eppendorfs, marked T0 to T10, containing 75 μl TE, 1 μl glycogen and 10 μl phenol.

3.	Combine 66 μl DNA from (1) (ie 10 μg), 50 μl 5X BAL-31 buffer and 133 μl H2O.  Equlibrate at 37 °C for 5 min then add 2 μl BAL-31 (5 U).

4.	Remove 25 μl aliquots (ca 1 μg) at 1 min intervals (T=0 to T=9) into tubes prepared in (2) above.

5.	Clean-up the reactions and finally resuspend in 18 μl of H2O.  Add 2 μl of second enzyme buffer, 5X excess of enzyme and digest for 1 h.

6.	Run gel of 5 μl of all samples to see which time intervals can be used to produce fragments of the desired size.

7.	Clean-up by the reactions and finally resuspend in 20 μl TE (found that the filling-in reaction was not necessary prior to ligation).

8.	Combine in a tube 4 - 8 μl insert (above), 2 μl vector (5 μg cut, genecleaned and resuspended in 30 μl TE) and water to a final volume of 10 μl.  Add 10 μl 2X ligation mix (1.25 U ligase per ligation).  Ligate for minimum of 2 h at room temp then transform 5 μl of the ligation to 100 μl competent cells and plate 200 μl expression mix on one X-gal plate.  Should get enough colonies to find all required deletions for the particular time slot.

9.	Miniprep to find shorts.  Minipreps need not be digested for first screening on gels.

10.	Maxiprep before sequencing.

Media, buffers and solutions

TE buffer

Bal31 buffer, 5x

Bal31 dilution/storage buffer


Recipe for Bal31 nuclease reaction buffer
  Recipes: BAL-31 reaction buffer (5X).

Contributor:

Zappe, H

Related:

Bal31 shortening

Storage:

4°C

Bal31 reaction buffer, 5x

Component 	stock conc	final conc.	/ 10ml

Tris-HCl, pH 8.0	1M	0.1M	1ml

CaCl2	1M	60mM	0.6ml

MgCl2	1M	60mM	0.6ml

EDTA, pH 8.0	0.5M	5mM	0.1ml

NaCl	5M	3M	6ml

Water	-	1.7ml


Recipe for Bal31 nuclease dilution/storage buffer
  Recipes: BAL-31 dilution/storage buffer

Contributor:

Zappe, H

Related:

Bal31 shortening

Storage:

-20°C

Bal31 dilution and storage buffer

Component 	stock conc	final conc.	/ 10ml

Tris-HCl, pH 8.0	1M	20mM	0.2ml

CaCl2	1M	5mM	50 μl

MgCl2	1M	5mM	50 μl

EDTA, pH 8.0	0.5M	1mM	20 μl

NaCl	5M	0.1M	0.20ml

Water	-	5.58ml

glycerol	44%(v/v)	4.40ml

All the components, except the glycerol, are combined in a 10 ml sterilin tube.  The tube is then filled to the 10 ml mark with sterile glycerol.  Mix by gently shaking the tube.  The volume marks on the sterilin tube are used as it is not possible to accurately pipette 5 ml of undiluted glycerol.


List of all contributors
  Contributors

Babb, Brendan

Coyne, Vernon

Jame, Di

Reid, Shez

Rybicki, Ed

Steyn, Lafras

Stutz, Helen

Zappe, Harold


Contributor: Zappe, Harold
  Contributor:

Dr Harold Zappe

Email address:

hzappe@medmicro.uct.ac.za

Comments:

Harold provided all the protocols for the first BBHelp.  These protocols were essentially harold's molecular biology techniques course.


Contributor: Babb, Brendan
  Contributor:

Brendan Babb

Email address:

bbabb@medmicro.uct.ac.za

Comments:

Brendan converted H Zappe's techniques course to Windows Help with some minor modifications.  This took him longer than anticipated with a fair amount of retyping text to fit a standard format.  Soon will be included the Microbiology department's techniques course, from V. Coyne, E. Rybicki, S. Reid and D. James...


Contributor: Steyn, Lafras
  Contributor:

Prof Lafras Steyn

Email address:

lsteyn@medmicro.uct.ac.za

Comments:

-


Contributor: Coyne, Vernon
  Contributor:

Dr Vernon Coyne

Email address:

Vernon@micro.uct.ac.za

Comments:

Vernon has put together the second edition of the Microbiology techniques manual, with the help of Ed Rybicki, Shez Reid and Di James.  This manual will be available as a printed document (contant Vernon directly).  [The first edition was done by Harold Zappe.]


Contributor: Rybicki, Ed
  Contributor:

Assoc Prof Ed Rybicki

Email address:

Ed@micro.uct.ac.za

Comments:

Ed's contributions were made through the Microbiology techniques manual.  See Vernon Coyne.


Contributor: Reid, Shez
  Contributor:

Dr Shez Reid

Email address:

Shez@micro.uct.ac.za

Comments:

Shez's contributions were made through the Microbiology techniques manual.  See Vernon Coyne.


Contributor: James, Di
  Contributor:

Di James

Email address:

Di@micro.uct.ac.za

Comments:

Di's contributions were made through the Microbiology techniques manual.  See Vernon Coyne.


Contributor: Stutz, Helen
  Contributor:

Helen Stutz

Email address:

Helen@micro.uct.ac.za

Comments:

Helen sent me the protocol for JAT preps (from Prof JA Thomson).


        BBHelp

On-line windows help for molecular biologists, including protocols, quick protocol guides, recipes and various useful appendices.

    About

    How to contribute

    How to use BBHelp

    Disclaimer

Protocols

Recipes

Appendices

Contributors


   Protocols

I.	Bacterial methods
DNA isolation, transformation, RNA isolation...

II.	DNA related protocols
Restriction, ligation, Southern blots, ...

III.	RNA related protocols
Northern blots, primer extension, ...

IV.	Protein related protocols
SDS-PAGE gels, Western blots, ...

V.	Polymerase Chain Reaction (PCR)
Descriptions and discussion of PCR techniques,


   Bacterial methods

A.	Competent cells

B.	Transformation

C.	Electroporation

D.	Total DNA isolation

E.	Plasmid perparation


   Bacterial

Competent cells

1.	Calcium chloride method

2.	DMSO method


   Bacterial

Transformation

1.	using Calcium chloride competent cells

2.	or DMSO competent cells


   Bacterial

Total DNA isolation

1.	DNA isolation (Zappe)


   Bacterial

Plasmid isolation

1.	Ish-Horowicz adaption plasmid preps (Zappe)

2.	JAT preps plasmid preps (Stutz)


   Bacterial

Electroporation

Not yet


  DNA methods

A.	Clean-up
before or after other reactions

B.	DNA ends
fill-in, nibble back, partial fill in, (de)phosphylation...

C.	DNA shortening
ExoIII, Bal31

D.	Electrophoresis
Agarose

E.	Labelling DNA probes

F.	Ligation

G.	Purification and precipitation
after labelling, ...

H.	Recovery
from agarose, ...

I.	Restriction endonuclease digestion

J.	Southern Blots

K.	Sequencing


  DNA methods

Electrophoresis

1.	Notes on agarose gel electrophoresis

2.	Resolution of DNA


  DNA methods

Clean up reactions

1.	Description (Zappe)

2.	Clean-up of DNA reactions (Zappe)

3.	Geneclean (Coyne)


  DNA methods

DNA shortening

1.	Heinekoff, ExoIII shortening

2.	BAL-31 shortening


  DNA methods

Labelling DNA

1.	Nick-translation (Zappe)


  DNA methods

Ligations

1.	Theory

2.	Table
size of DNA vs amount to use

3.	Protocol descriptions

4.	Ligation in agarose

5.	Notes on two reaction types

6.	Hints for troubleshooting (Coyne)


  DNA methods

Recovery of DNA

1.	Direct elution

2.	Electroelution

3.	Geneclean


  DNA methods

Purification and precipitation of DNA

1.	Spin column purification (Zappe)

2.	Spin column purification (Coyne)

3.	Cesium chloride
gradient centrifugation for DNA separation

4.	Ammonium acetate/ethanol precipition

5.	Sodium perchlorate/Isopropanol precipitation


  DNA methods

Restriction endonuclease digestion

1.	Digestion (Zappe)

2.	Restriction enzyme sites


  DNA methods

Southern hybridization

1.	radiolabelled probe method (Zappe), entire protocol is subdivided into steps.

2.	Gel prep and transfer method (Coyne)

3.	DNA-DNA hybridization by modified Church method (Coyne)


  DNA methods

Manipulation of DNA ends

1.	Fill-in
3'-recessed (5'-overhang) fill-in with Klenow

2.	Flush-ending
(nibble-back)

3.	Partial fill-in

4.	Dephosphorylation of DNA (description)

5.	CIPping vector DNA (Coyne)


  RNA methods

sorry, none yet


  Protein methods

A.	SDS-PAGE
SDS Polyacrylamide electrophoresis of proteins

B.	Western Blot
Antibody detection of specific proteins


  Protein methods

SDS Polyacrylamide electrophoresis of proteins

1.	The protocol of Zappe

2.


  Protein methods

Western blots of proteins

1.	General description

2.	BM protocol (Zappe)


   Appendix

Plasmid maps

1.	pSK Bluescript

2.	pUC19

3.	pBR322


   Appendices

A.	Genetic code

B.	Plasmid maps

C.	Ice and cooling mixtures

D.	Nucleic acid weight data

E.	List of all references

F.	λ (Lambda) DNA standards

G.	Radioactivity

H.	Restriction enzymes

I.	Amino acid codes

J.	Partial fill-in sites and restriction enzymes


List of all recipes
  Recipes

Please look up entries in alphabetical order of their full names.  Where not otherwise stated, storage is at room temperature

A

acrylamide gel mix

Acrylamide stock

Ammonium acetate

Ammonium persulphate

Ampicillin

ATP

B

Bal31 dilution/storage buffer

Bal31 reaction buffer

Blocking buffer stock solution, Westerns

Blocking buffer, Westerns

Blot buffer, Westerns

BSA (bovine serum albumin)

C

Calcium chloride, CaCl2

Cell Suspension buffer

Chloroform-isoamyl alcohol mix (24:1)

Church hyb buffer

Church prehyb buffer CHB

CTAB/NaCl (hexadecyltrimethyl ammonium bromide)

Cysteine

D

Denaturation solution 2 (0.4M NaOH/1M NaCl)

Destain, SDS-PAGE

Dithiothreitol, DTT

DNA digestion: core reaction buffers

DNA digestion: enzyme dilution buffer

E

EDTA

Ethanol

Ethidium bromide, EtBr

ExoIII reaction buffer

F

G

Gel mixes, SDS-PAGE

Glucose

H

Hybridization solution (Zappe)

I

IPTG, Isopropyl-ß-D-thio-galactopyranoside

Isopropanol, salt-saturated

J

K

Klenow buffer (10x)

Klenow mix

L

Laural sulphate, see sodium dodecyl sulphate

Ligation dilution buffer

Ligation mix

Ligation reaction buffer

Luria broth, LB

Lysing solution

M

Magnesium chloride, MgCl2

Magnesium sulphate, MgSO4

Minimal medium, E. coli

Minimal medium 5x salts, E. coli

Mungbean nuclease dilution/storage buffer

Mungbean nuclease reaction buffer

N

O

P

PB (phosphate buffer)

Phenol Chloroform mix

Phenol (buffered pH 7.6, 10mM Tris)

Potassium chloride

Potassium salts

Q

R

Resolving buffer, SDS-PAGE

RNAse mix

S

S1 mix

S1 stop mix

Salt, see sodium chloride

Sample buffer, SDS-PAGE

SDS, see sodium dodecyl sulphate

Sephadex G-50

Sma I reaction buffer

Sodium acetate, NaAc

Sodium chloride, NaCl

Sodium dodecyl sulphate, SDS

Sodium hydroxide, NaOH

Sodium perchlorate, NaClO4

Solution III, plasmid preps

Solution II, plasmid preps

Solution I, plasmid preps

SSC (salt sodium citrate) 20x

ST1  Southern transfer solution 1

ST2  Southern transfer solution 2

ST3  Southern transfer solution 3

Stacking buffer, SDS-PAGE

Stain, SDS-PAGE

STE

T

TA buffer

TAE buffer,Tris-acetate-EDTA

Tank buffer, SDS-PAGE

Taurine buffer (TTE)

TB buffer

TBE buffer

TE buffer, Tris-EDTA buffer

TEMED

Terrific broth

Tracking dye (IV)

Tracking dye (Coyne)

Tris buffer

Tris-Base

tRNA

TSB, TSBG

U

V

W

Wash buffer A (WBA)

Wash buffer B (WBB)

Wash buffer C (WBC)

X

X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside

Y

YT 2x, yeast tryptone medium

Z

Zinc acetate

Zinc sulphate


Clean-up DNA reactions, description
  DNA: clean-up

Related methods

Description:

This set of steps is followed to remove enzymes and change buffers between different reactions.  Before using this protocol, check the reaction conditions of the next enzyme you plan to use - it may work in your current buffer.


Clean-up DNA reactions, protocol
  DNA: clean-up

Related       Description       DNA reactions

Contributor:

Zappe, H

Reference:

-

1.	Make up the volume of the reaction to 90 μl (or some suitable volume) with TE.

2.	If the quantity of DNA is less than 5 μg, add 1 - 2 μl of BM glycogen.  This addition will not affect any further reactions using the DNA and simply aids precipitation of small quantities of DNA.

3.	Add 10 μl (or 1/10 volume) of phenol and emulsify by shaking.

4.	Add 1 volume of CHCl3/isoamyl alcohol (24:1) and again emulsify by shaking.

5.	Spin for 5 min and recover top aqueous phase

6.	Add 1/10 volume of 5 M NaClO4 and an equal volume of isopropanol.  Mix, leave on ice for 5 min and then spin for 10 min.

7.	Wash the pellet with 70 % EtOH and spin 2 min with tube re-orientation.  Pour off EtOH, spin to pellet the last drops and draw off with a P200 pipette.

8.	Resuspend in water or TE depending on the following reaction.

Media, buffers and solutions	Form

TE buffer

NaClO4

chloroform:isoamyl alcohol

EtOH

Phenol


Heinekoff shortening of DNA fragments, description
  Heinekoff shortening

Protocol       Related

Description;

Shortening of DNA using ExoIII nuclease.  Insert to be shortened must have two correct types of r/e sites on side of insert to be shortened.  One with a 3' overhang to protect and one that is blunt or 5' overhang to cut back on.

To see what enzymes have which overhangs, see Restriction enzyme sites.  The enzymes with negative  values for the overhang are protected, eg KpnI with overhang of -4.


Heinekoff shortening of DNA fragments, protocol
  Heinekoff shortening

Related       Description        R/E sites

Contributor:

Zappe, H

Reference:

-

1.	Digest 12 μg DNA with appropriate enzymes: one 5' prime overhang and one 3' to protect the vector.  As the sites in the MCS are close together, cut 6 μg with each of the enzymes, check for cutting on a minigel, then mix digestions and add a little more of each enzyme (2 X excess).

2.	Precipitate and resuspend in 100 μl Exo III buffer

3.	Prepare 10 tubes with S1 mix (20μl per tube) and hold on ice.  Label them 0, 20, 40, 60, etc. for time of treatment.

4.	Equilibrate DNA at 37°C (5 min), and remove 1st 9 μl sample (T=0) to an S1 tube.

5.	Add 300 U Exo III to remaining DNA, mix quickly!.

6.	Remove 9 μl samples from the tube kept at 37°C at 20 s intervals to the respective S1 tubes and mix well.

7.	Raise S1 mixes to room temp and leave for 30 min.

8.	Add 3.5 μl S1 stop and place at 70°C for 10 min.

9.	Remove 6 - 8 μl from every 2nd or 3rd fraction and run gel (0.7 % - as fragment is not released from the vector as with BAL-31 shortening) and check shortening.

10.	Add 3 μl Klenow mix to each tube and 1 μl klenow enzyme and leave for 3 min at room temperature (to fill-in any sticky ends).

11. Add 1 μl dNTP's (0.125 mM/μg DNA)/tube and leave 5 min at room temperature.

12. Add 100 μl ligation mix.

13.	Ligate 1 - 3 h at room temperature and transform cells eg LK111: 10 μl ligation + 50 - 100 μl cells.

14.	Miniprep and restriction analysis to find resections.

Media, buffers and solutions	Form

ExoIII buffer

S1 mix

Klenow mix

S1 stop

ligation mix

dNTP's


Recipe for S1 stop
  Recipes: S1 stop

Contributor:

Zappe, H

Related:

Heinekoff shortening

S1 stop

Component 	stock conc	final conc.	/ 100 ml	/1l	/10ml

Tris base	0.3M

EDTA	10mM

Water	-	to 100 ml


Recipe for making ExoIII buffer
  Recipes: ExoIII buffer

Contributor:

Zappe, H

Related:

Heinekoff shortening

ExoIII buffer

Component 	stock conc	final conc.	/ 10 ml	/1l	/10ml

Tris-HCl	1M	50mM	100 μl

MgCl2	1M	5mM	50 μl

Water	-	9.45 ml


Recipe for S1 mix
  Recipes: S1 mix

Contributor:

Zappe, H

Related:

Heinekoff shortening

S1 mix

Component 	stock conc	final conc.	/ 10 aliquots

S1 buffer, 10x	10x	1x	27 μl

water	172 μl

S1 nuclease 	60U-	1 μl


Recipe for klenow mix
  Recipes: Klenow mix

Contributor:

Zappe, H

Related:

Heinekoff shortening

Klenow mix

Component 	stock conc	final conc.	/ 10 aliquots

Tris-HCl, pH 7.6	1M	0.1M	6 μl

MgCl2	1m	3 μl

water	21 μl


Recipe for ligation mix
  Recipes: ligation mix

Contributor:

Zappe, H

Related:

Heinekoff shortening

ligation mix

Component 	stock conc	final conc.	/ 10 aliquots

ligation buffer 	10x	1x	125 μl

water	870 μl

ligase 	25 U-	25 μl


DNA isolation from bacterial cells, description
 DNA isolation from bacterial cells

related        protocol

Description:

A.	The method of Zappe is quite wordy and applies to most bacteria
protocol

B.	Large scale bacterial genomic DNA prep by Coyne
protocol


DNA isolation from bacterial cells, protocol
 DNA isolation from bacterial cells

related        description

Contributor:

Zappe, H

Reference:

Based on the method of Marmur (J. Mol. Biol. 3:208-218, 1961).  Modified by J.L. Johnson (Virginia Tech.)

The volumes of buffers given are for cultures of 300-600 ml of E.coli type cultures (good growers).  Reduce volumes for low density or smaller cultures.  Note that mycobacteria may have a similar culture density, but DNA yield would be much lower due to the cell envelope.

1.	Harvest the cells by centrifugation at 7500 RPM for 10 min.  Decant the supernatant and allow the centrifugation bottles to drain upside down on paper towel.  Do this under appropriate conditions for pathogens.  Resuspend the cells in 20 ml of suspension buffer for enzymatic lysis or 10 - 20 ml for disruption by French pressure cell treatment.  See 2cii for glass bead method.  The last two methods require a thin paste of cells - not too dilute.  Resuspend the cells properly, either by vortexing the pellet with 1 or 2 ml buffer before adding the rest of the buffer, or by drawing up and expelling the cell/suspension buffer mixture with a 10 ml pipette.  There should be no clumps of cells after this step.  Transfer the cells to a suitable flask (125 - 150 ml).

2.	Digest the cells with lytic enzyme.

2a.	Gram-negative bacteria.  Add dry lysozyme (4 mg/ml or about 1/8 teaspoon for 25 ml!) and incubate.  The time and temperature can vary from 10 min at room temperature to 4 h at 37°C depending on the organism.  Remove small samples (100μl) periodically and mix with 100μl lysing solution.  If the cells lyse, go to step 3, if not continue incubation.  More lysozyme can also be added.

2b.	Gram-positive bacteria.  Add dry lysozyme (8 mg/ml or about 1/4 teaspoon per 25 ml!) and incubate at 37°C.  Start doing the lysis test from 15 min onwards.  Other enzymes are also available if lysozyme does not work, but lysozyme is the cheapest.

2c.	Recalcitrant bacteria.  Some organisms will not be made sensitive to detergent lysis using lytic enzymes and must be physically disrupted.  Either of the following methods can be used.

i.	French Pressure cell.   Pass the cell suspension through the French Pressure cell at 16 000 psi (upper limit for many cells) into an equal volume of lysing solution.

ii.	Glass beads.  Using  a Braun homogeniser with liquid CO2 cooling.  Scrape the cell pellet form the centrifugation bottle and place it into a tared Braun shaker bottle.  Make up the weight to 20 g with suspension buffer (cells + buffer), add 20 ml glass beads (0.1 mm diameter) and shake for 5 min at 4000 cycles per minute (use the cooling system).  Separate the lysate from the glass beads by filtration through a coarse 60 ml scintered glass filter.  Use 20 ml lysing solution to wash the last of the lysate from the beads.

3.	Add an equal volume of the 2X lysing solution (unless its already been added in step 2c i or ii above) and a volume of 5 M NaClO4   equal to 1/4 of the combined total.  This mixture forms a precipitate - heat to 50°C to get it back into solution.  If the cells were susceptible to lysis, mix with a swirling action to get a uniform mixture before all the cells lyse.  Lysis is evident by the turbid solution becoming translucent and very viscous.  Incubate the lysate for 4 h at 50 - 60°C to degrade cellular proteins.  As little as 1 h may be OK, but overnight is sometimes convenient and better.

4.	Add 15 ml phenol-chloroform solution, shake by hand to form a uniform emulsion and then shake on a wrist action shaker for 20 min.  Transfer the mixture to a 50 ml centrifuge tube and centrifuge at 12 000 RPM for 10 min at room temperature.  Meanwhile rinse  the flask and let it drain.  Slowly decant as much of the upper aqueous layer back into the flask as you can.  Do not allow any of the organic phase to go over.  The remainder of the aqueous phase can be collected by using an inverted 2 or 5 ml glass pipette and a pi-pump.  Do not use narrow bore pipettes or Gilson tips as significant shearing of the DNA can occur.  Repeat the extraction procedure twice more.  Extraction of proteins is complete if there is little or no white precipitate at the interface after the centrifugation step.

5.	Carefully decant the aqueous phase from the last phenol-chloroform extraction into a suitable flask (125 ml Erlenmeyer).  Taring the flask and then weighing the solution is a good way of estimating the volume (1 ml = 1 g).  Add 0.6 volume of Isopropanol and swirl to mix.  The nucleic (DNA and RNA) will precipitate and form a loose clot (unless the cells were physically disrupted, where it will be necessary to centrifuge the solution).  Hold the clot back with a Pasteur pipette and pour off the lysate-Isopropanol solution.  Add 25 ml 76 % ethanol and allow to stand for 10 min.  Decant the ethanol and repeat the wash.  Decant the ethanol and press the clot of nucleic acid with a Pasteur pipette to get rid of most of the ethanol.  Allow the precipitate to dry at 37°C for 15 min.  Never centrifuge preparations of high Mol. wt. DNA - they take a very long time to resuspend (days).

6.	Dissolve the nucleic acid precipitate in 20 ml TE buffer (may take a few hours), add 0.25 ml of RNase mix and incubate at 37 °C for 1 h (Note that in any nucleic acid preparation, most of the nucleic acid at this point is RNA).  Extract the solution once with 5 ml chloroform:isoamyl solution, centrifuge and save the aqueous layer.

7.	Add 0.1 volumes of 3 M Sodium acetate, mix, overlay with 2 volumes of 95% ethanol, and collect the DNA by spooling onto a glass rod (or Pasteur pipette) by rotating the rod and stirring the solution at the same time.  Continue until both phases are completely mixed.  Alternatively, the DNA can be precipitated just by swirling the solution as in 5 above.  Collect the "clot" of DNA, wash and dry the precipitate as in 5 above.

8.	Dissolve the DNA in 3 - 5 ml TE (again, this could take a day - leave at 4°C).  Do a 310 - 220 nm scan to determine the actual concentration.  Store at a concentration of at least 0.5 mg/ml at -20°C.

Note:	The procedure can be stopped and the DNA solution stored at 4 °C for up to a few days anyhwere after step 4.  However, do not store solutions containing phenol - decant the aqueous phase and store this.

Media, buffers and solutions    	Conc	Form	Storage

Cell suspension buffer	1x	liquid stock

Lysing solution	2X	liquid stock

NaClO4 	5M

Isopropanol

Ethanol

sodium acetate	3M

TE buffer

Chloroform:Isoamyl mix	(24:1)

Phenol-Chloroform mixture

RNase mix

Note: If the cells are going to be physically disrupted (French press of glass beads), then do not add the sucrose to the cell suspension buffer.


DNA manipulation, filling in of 5' overhangs, description
  DNA: Filling-in 3'-recessed ends of DNA

Protocol       Related

Description:

Filling-in 3'-recessed (i.e. 5' overhang) ends of DNA.


DNA manipulation, filling in of 5' overhangs, protocol
  DNA: Filling-in 3'-recessed ends of DNA

Description       Related

Contributor:

Zappe, H

Reference:

-

1.	After clean-up of the digest resuspend 1 - 5 μg DNA pellet in 32 μl  water, 4 μl of 10 X Klenow buffer and 4 μl 10 X dNTP solution (5 mM mix i.e. each at 1.25 mM).

2.	Add 1 - 2 U Klenow and incubate at 37 °C for 15 min.

3.	Clean-up before continuing.

Media, buffers and solutions	Form

Klenow

Klenow buffer, 10x

dNTP, 5mM mix (each at 1.25mM)


Recipe for klenow buffer
  Recipes: Klenow buffer, 10x

Contributor:

Zappe, H

Related:

DNA reactions        Klenow fill-in

Storage:

-20°C

Klenow buffer, 10x

Component 	stock conc	final conc.	/ 10 ml

Tris-HCl, pH 7.6	1M	0.1M	1ml

MgCl2	1M	0.1M	1ml

NaCl	5M	0.5M	1ml

DTT	0.5M	10mM	0.2ml

BSA	20mg/ml	0.5mg/ml	.25ml

Water	-	6.55 ml


DNA, flush ending overhangs with mung bean nuclease, description
  DNA: Flush ending of overhangs

Protocol       Related

Description:

using Mung Bean nuclease.  Method from New England Biolabs catalogue

Filling-in 3'-recessed (i.e. 5' overhang) ends of DNA.


DNA, flush ending overhangs with mung bean nuclease, protocol
  DNA: Flush ending of overhangs

Description       Related

Contributor:

Zappe, H

Reference:

New England Biolabs catalogue

1.	DNA to be treated at a concentration of about 1 μg/10 μl reaction (5 μg in a 50 μl reaction with 5 U nuclease).

2.	Add 0.5 - 1 unit mung bean nuclease per μg DNA.

3.	Incubate 37°C for 30 min.

4.	Clean-up the reaction.

Media, buffers and solutions	Form

Mungbean reaction buffer

Mungbean dilution/storage buffer


Recipe for Mungbean reaction buffer
  Recipes: Mungbean reaction buffer, 10x

Contributor:

Zappe, H

Related:

Flush ending

Storage:

4°C

Mungbean nuclease reaction buffer, 10x

Component 	stock conc	final conc.	/ 10 ml

Sodium acetate, pH 5.0	1M	0.5M	5ml

NaCl	5M	0.3M	0.6ml

ZnSO4	0.1M	10mM	1ml

Water	-	to 3.4ml


Recipe for Mungbean dilution/storage buffer
  Recipes: Mungbean dilution/storage buffer, 10x

Contributor:

Zappe, H

Related:

Flush ending

Storage:

-20°C

Mungbean nuclease dilution/ storage buffer, 10x

Component 	stock conc	final conc.	/ 10 ml

Sodium acetate, pH 5.0	1M	0.01M	0.1ml

Zn-acetate	0.1M	0.1mM	10 μl

Cysteine	0.1M	1mM	0.1ml

Triton X100	0.1%(v/v)	0.001%	0.1ml

Water	-	to 4.69ml

glycerol	50%(v/v)	5ml

All the components, except the glycerol, are combined in a 10 ml sterilin tube.  The tube is then filled to the 10 ml mark with sterile glycerol.  Mix by gently shaking the tube.  The volume marks on the sterilin tube are used as it is not possible to accurately pipette 5 ml of undiluted glycerol.


Recipe for zinc sulphate
  Recipes: Zinc sulphate

Contributor:

Zappe, H

Related:

-

Zinc sulphate

Component 	stock conc	final conc.	/ 10 ml

Zinc sulphate	-	to 4.69ml

water	5ml


Recipe for zinc acetate
  Recipes: Zinc acetate

Contributor:

Zappe, H

Related:

-

Zinc acetate

Component 	stock conc	final conc.	/ 10 ml

Zinc acetate

water	50%(v/v)	5ml


Recipe for cysteine
  Recipes: Cysteine

Contributor:

Zappe, H

Related:

-

Cysteine

Component 	stock conc	final conc.	/ 10 ml

Cysteine	0.1M	1mM	0.1ml

Water	-	to 4.69ml


Notes on agarose gel electrophoresis (Zappe)
  Agarose gel electrophoresis
Related        λ-(Pst)        Gel resolution        λ standard pics

Contributor:

Zappe, H

Notes:

1.	We use the standard submerged gel system as described by Sambrook et al.

2.	Generally either TAE or TB is the preferred buffer.

3.	Agarose concentrations vary from 0.6% (w/v) (for large fragment separation to 2% (w/v) for small fragments.

4.	Using a 254 nm lightbox, 40 - 50 ng of DNA per band is clearly visible.  For any routine plasmid digest (up to about 6 fragments), 200 ng is plenty to see all the bands.

5.	Fragment sizes are calculated by extrapolation from a standard curve of the mobility of lambda DNA fragments.  The particular lambda digest is chosen to suit the fragment sizes generated.  Routinely use lambda PstI, and never run a gel without a standard!

Media, buffers and solutions

TAE buffer

TB buffer


Protocol for standard DNA size markers, Lambda-PstI digest
  λ-PstI digest

Related (gels)        Electrophoresis notes      λ-standard pics

Contributor:

Zappe, H

Reference:

-

1.	Combine 200 μl lambda DNA, 100 μl 100 restriction buffer and 700 μl water in an eppendorf.  Mix and divide into 5 X 200 μl.  Add 20 U PstI to each tube and incubate 1 - 2 h at 37 °C.  Add 40 μl tracking dye to each tube.  Load 15 - 20 μl per gel lane as standards.

Restriction fragments (in bp) generated by PstI in lambda DNA

Frag	size	Frag	size	Frag	size
1	11497	11	1700	21	216
2	5077	12	1159	22	211
3	4749	13	1093	23	200
4	4507	14	805	24	164
5	2838	15	514	25	150
6	2560	16	468	26	94
7	2459	17	448	27	87
8	2443	18	339	28	72
9	2140	19	264	29	15
10	1986	20	247


Recovery of DNA from agarose gels, discussion
  Recovery of DNA from agarose gels

Related methods

Contributor:

Zappe, H

Discussion:

Fragments of DNA resolved of agarose gels can be recovered and cloned for further experimentation.  A variety of methods have been described including electroelution (various methods using dialysis tubing or charged paper such as DEAE), freeze and squeeze and others.

A number of kits such as GeneClean are also available for this purpose.  They use the principle that DNA binds to chemically modified powdered glass under certain buffer conditions and can be eluted by a salt concentration change.  Yields of about 90% are obtainable.

Another commomly used method is the use of Gel-ase, an enzyme that digests the gel, leaving the DNA in solution.  Yield by this method is very high.

Cloning methods have also been described using low melting point agarose, where it is not necessary to remove the agarose at all (see Rapid cloning method).


DNA recovery by direct band elution
  DNA recovery: direct band elution

Description        Related

Contributor:

Zappe, H

Reference:

Rapid and simple purification of PCR products by direct band elution during agarose gel electrophoresis.  H Hansen and H Lemke (1993), Biotechniques 14(1):28-29.

1.	Cast the gel with two sets of wells (primary and trap), the distance apart depends on the size of the fragment to be recovered.

2.	Load the samples into the primary wells and run the gel so that the samples migrate towards the trap wells.  When the DNA band of interest is about 0.5-1.0 cm from the trap well, fill the well with running buffer containing 10% (v/v) 3 M sodium acetate and 10 % sucrose.  This lowers the pH of the running buffer in the area around the trap well and serves to focus the approaching DNA band.  The migration is monitored as the band moves into the well, and the DNA sample can then be recovered by a pipette.

3.	Precipitate the DNA by the addition of glycogen and 1 volume of isopropanol as usual, and resuspend the pellet in the buffer of choice.

Media, buffers and solutions

glycogen

sucrose. 10%

sodium acetate 3M


Recipe for Tris-borate buffer
  TB buffer

Contributor:

Zappe, H

Related:

Gel electrophoresis

Tris-borate buffer (5x)

Component 	stock conc	final conc.	/ 1000ml

Tris-base	54g

Boric acid	27.5g

EDTA, pH 8.0	0.5M	20ml

Do not store Tris-borate in a glass bottle.


Recipe for gel tracking dye (IV), 6x
  Gel tracking dye IV

Contributor:

Zappe, H

Related:

Gel electrophoresis

Gel tracking dye IV (6x)

Component 	stock conc	final conc.	/ 10ml

Bromophenol blue	0.25%(/v)	25mg

sucrose	40%(w/v)	4g

EDTA, pH 8.0	0.5M	20mM	0.4ml

water	to 10ml


DNA resolution of DNA fragments in agarose
  Agarose gel electrophoresis
Related

Contributor:

Babb, B.

Reference:

New England Biolabs catalog (1992), p209

Percentage gel versus optimum resolution for linear DNA (kb)

% Gel	DNA (kb)

0.5	30 to 1.0

0.7	12 to 0.8

1.0	10 to 0.5

1.2	7 to 0.4

1.5	3 to 0.2kb


DNA, Ligations in agarose
  DNA: Ligations in agarose

Protocol       Ligations       Electrophoresis

Description:

Cloning involves restriction of the vector and insert DNAs, inactivation of the enzymes, usually by phenol or heat, and then DNA precipitation (DNA clean-up).  These manipulations can result in considerable DNA loss and sometimes unpredictable concentrations.  When more manipulations are involved such as filling-in, CIPing, BAL31 etc. the situation only gets worse.  In most cases, only highly purified plasmid DNA (CsCl gradients) can be used as miniprep DNA contains too many impurities for some of the more sensitive reactions.

A method is presented whereby the restricted (filled-in, CIPped etc) DNA is electrophoresed (and thus purified of enzymes, linkers, unwanted fragments etc) in low melting point (LMP) agarose and the DNA bands of interest (insert and vector) are cut out, melted, combined and ligated without the need to remove the gel by phenol extraction.

Advantages of gel based manipulations

1.	Very quick - can subclone many fragments per day.

2.	No loss of DNA, no DNA clean-up required.

3.	Incomplete digestion is no problem - simply remove only the right band.

4.	DNA in the gel can be melted and used n times.

5.	Digestions of specific restriction fragments can be performed in the same way by adding 2X restriction mix.

6.	Miniprep DNA can be used for all manipulations.


DNA, Ligations in agarose
  DNA: Ligations in agarose

Description       Related

Contributor:

Zappe, H

Reference:

-

1.	If at all possible, cut insert and vector with 2 enzymes to prevent formation of parentals.

2.	Run mini LMP gel (0.5-1.5%) using SeaPlaque® agarose in Tris-Acetate buffer (50 mM, pH 8.2 no EDTA, no EtBr).

3.	Stain gel, view at 310 nm and cut out required bands in as small a vol. as possible (30 - 50 μl)

4.	Melt slices at 70 °C for 5 min, equilibrate at 37 °C, and then combine at required ratios in a total vol of 10 µl.

5.	Add 10 μl 2X ligation mix (containing 0.1-0.25 U ligase/10 μl (sticky) or 1u/10 μl (blunt ligation).

6.	Hold at RT for 2 h, then melt at 70 °C as before and dilute with 5 vol 0.1M CaCl2.

7.	Transform 10 μl with 100 μl of comp. cells, and finally plate 200 μl on 1 plate (with pUC, 1 plate gives plenty of colonies (ca. 200) and saves on X-gal).

Troubleshooting

1.	The bigger bands in any DNA gel lane always contain a small % of the fragments below - if the smaller bands have the same ends, expect to find some clones containing these fragments, or use biochemical selection.

2.	The only enzyme that does not work in gel containing solutions is T4 polynucleotide kinase.

Media, buffers and solutions

TA buffer

ligation mix

X-gal


Recipe for TA buffer
  Recipes: TA buffer

Contributor:

Zappe, H

Related:

Gel based DNA manipulation

TA buffer

Component 	stock conc	final conc.	/ 10ml

Tris-HCl, pH 8.0	1M

NaAcl	5M

Water	???-	5.58ml

??


About BBHelp

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BBHelp is designed for people already familiar with some molecular biology techniques.  It makes information on protocols, recipes, plasmid maps, etcetera, accessible in a quick, concise and pertinent manner.

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BBHelp is being maintaining and updated by Brendan.  Incorporated into BBHelp, is the U.C.T. Medical microbiology honours techniques course manual of Harold Zappe, and the U.C.T. Microbiology techniques manual of Vernon Coyne, Ed Rybicki, Shez Reid and Di James.  Thanks to Lafras Steyn for supporting the generation of BBHelp.  See contributing authors for a complete list of contributors.

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DNA ligation theory (Zappe)
  DNA ligations

Related        Table         Descriptions

Contributor:

Zappe, H

Theory:

A good overview of ligation theory is presented in Sambrook et al. pages 1.63-1.67.  Essentially there are two types of reactions that one wants to achieve;  namely limited concatermerisation and recircularisation, and they are mutally exclusive, so some form of compromise must be used to achieve ligation.  The ratio of recircularised to concatermeric ligation products is dependent on two factors i and j.

j =	the effective concentration of one end of a DNA molecule in the neighbourhood of the other end of the same molecule.

i = 	a measure of all complementary terminii in the solution.

where j = i	there is an equal chance of one end making contact with a different end or the other end of the same molecule.

where j > i	higher chance of recircularisation.

where j < i	higher chance of concatermerisation.

A theoretically maximum yield of chimeric molecules (ie. 1 vector and 1 insert molecule recircularised) is obtained where j:i = 3 for the plasmid concentration and the ratio of plasmid to insert DNA is _ 1.  DNA concentrations in ligations should be caculated as in the following example:

Using the vector pUC18, and for 1 < j:i < 3, the ligation should contain about 20 - 60 µg of DNA per ml.  If this concentration is converted to pmoles/ml, then the formula can be applied to any vector and insert.  In practice 10 pMoles per ml (18 μg of pUC18) works well, but this can be increased to about 30 pMoles/ml (54 μg of pUC18).  The insert concentration would be in the 30 - 90 pMole range.  Note that you cannot simply mix DNA fragments based on μg quantities; it is essential that molar ratios be combined.

The relationship of MW or Mr and kb of DNA.

1 kb of DNA has an approximate Mr of 0.662 MDaltons (10^6)

therefore	1 pmol DNA = (0.662 X kb) μg

or	1 pmol DNA = (MW)(expressed as Mega Daltons) μg.

For example:	 1 mole of 1.5 kb = (0.662 X 1.5) = 1 X 10^6 g

1 picomole 	= 1 X 10^6 X 10^-12 g

= 1 X 10^-6 g (which is μg)

therefore 	1 pMole of pUC18 	= 2.686 X 0.662

= 1.8 µg DNA

==>	Accordong to theory, use about 20 μg or 12 pmole DNA per ml ligation and use about  double to 3 times the amount of insert at about 25 - 35 pmole / ml

Ligation volumes are never in excess of 0.1 ml; generally the volume is between 10 and 50 μl.  Therefore for a 20 μl ligation one would combine 0.4 μg (pUC 18) vector and 1.66 μg of a 5 kb insert.

Simplified concentation calculations

A simplified way of combining molar ratios of vector and insert is by using ratios of size. For example, for a vector of 3 kb and an insert of 1 kb, the quantity of insert used would always be 1/3 that of the vector for a 1:1 molar ratio.

1.	for a 1:1 (vector:insert) molar ratio, combine 3 μg of vector and 1 μg of insert.

2.	for a 1:3 (vector:insert) molar ratio, combine 3 μg of vector and 3 μg of insert.

For pUC18, use about 20 μg/ml DNA (11 pmole) or 400 ng per 20 μl ligation.

Improving the formation of recombinant molecules.

1.	Wherever posible use 2 different enzymes in cloning experiments to prevent vector religation and insert circularisation.  Referred to as forced cloning.

2.	When cutting a vector with 2 enzymes, a small "stuffer" fragments is cleaved out.  If this remains present in the ligation, it can be re-inserted thus reducing the number of useful clones.  Gel purify the vector form this fragment.

3.	If only one restriction enzyme site is available, use an insert:vector ratio of 2:1 or 3:1 as described above.

4.	Use biochemical selection to prevent cloning of unwanted fragments - i.e. cut the unwanted fragments with a second enzyme that does not cut the fragment of interest (saves having to gel purify fragments).

5.	The New England Biolabs (NEB) Transcript (Vol 1 No. 1) gave a tip on improving blunt-end ligation.  Reduce the ATP concentration in the ligation buffer to 0.5 mM (20 X less) giving a final reaction concentration of 0.05 mM).  Therefore add 5 µl of 0.1 M ATP and an extra 95 µl of water to the buffer given below.

6.	General formulae for the calculation of pmol ends/µg:

1.  For circular molecules: 2N X 10^6   pmol ends/µg

MW

2.  For linear molecules:  2(N+1) X 10^6  pmol ends/µg

MW

where  N = the number of restriction sites

MW is in Daltons (e.g. pBR322 = 2.9 X 10^6)


Table of DNA concentrations to use in ligations
  DNA length versus amount

Related

Contributor:

Babb, B

Table:

Table of DNA concentrations to use in ligations :

The amount of DNA in ng required in order to get 5pmol concentration of the DNA of a particular length in a particular reaction volume.  eg you need 20ng of a 0.6kb fragment in 10μl to have 5pmol.

Length (kb)	5pmol 	ng DNA for a	ng DNA for a

(μg/ml)	10μl reaction	20μl reaction

0.1	0.331	3.3	6.6

0.2	0.662	6.6	13.2

0.3	0.993	9.9	19.9

0.4	1.324	13.2	26.5

0.5	1.655	16.6	33.1

0.6	1.986	19.9	39.7

0.7	2.317	23.2	46.3

0.8	2.648	26.5	53.0

0.9	2.979	29.8	59.6

1.0	3.310	33.1	66.2

1.5	4.965	49.7	99.3

2.0	6.620	66.2	132.4

2.5	8.275	82.8	165.6

3.0	9.930	99.3	198.6

3.5	11.585	115.9	231.7

4.0	13.240	132.4	264.8

5.0	16.550	165.5	331.7


Recipe for ligation reaction buffer
  Recipes: Ligation reaction buffer

Contributor:

Zappe, H

Related:

DNA ligations

Storage:

-70°C

Generally manufacturers supply a ligation buffer, but a tried and tested buffer is given below.

Ligation reaction buffer, 10x

Component 	stock conc	final conc.	/ 1ml

Tris-HCl, pH 7.6	1M	66mM	0.66ml

MgCl2	1M	6mM	66 μl

DTT	-	?	15.4mg

ATP	0.1M	10mM	100 μl

Water	-	174 μl

This buffer is stable for more than 2 years when stored at at -70 °C  The ATP is made according to Sambrook et al.(pH 7.0).


Recipe for ligation dilution buffer
  Recipes: Ligation dilution buffer

Contributor:

Zappe, H

Related:

DNA ligations

Storage:

-20°C

Ligation dilution buffer, 10x

Component 	stock conc	final conc.	/ 10ml

Tris-HCl, pH 7.6	1M	20mM	0.2ml

EDTA	0.5M	1mM	2 μl

DTT	0.5M-	5mM	10 μl

KCl	1M	60mM	0.6ml

water	4.8ml

glycerol	44%(v/v)	4.4ml

The buffer is made according to the following table and stored at -20 °C:


Recipe for potassium chloride
  Recipes: Potassium chloride

Contributor:

Zappe, H

Related:

-

Potassium chloride (KCl), 1M

Component 	stock conc	final conc.	/ 1l

KCl	-	1M	??g

water	up to 1l


Protocols for DNA ligation
  DNA ligation protocol

Related        Theory

Two general ways of doing:

Method A

Set up two times ligation mix and add to all tubes

Method B

Do each tube independently


DNA, setting up ligation reactions with ligation mixes
  DNA ligation: ligation mixes (A)

Related       Descriptions

Contributor:

Reference:

1.	Mix up 2x ligation mix

2.	add dna and water to tubes in 10 μl

3.	add 10 μl ligation mix to each tube

Media, buffers and solutions

Ligation buffer

Ligase dilution buffer

Ligation mix


DNA, setting up ligation reactions
  DNA ligation protocol (B)

Related       Descriptions

Contributor:

Reference:

???

Media, buffers and solutions

Ligation buffer

Ligase dilution buffer


Notes on DNA ligation
  DNA ligations - Notes

Related        Descriptions

Contributor:

Coyne, V

Notes:

A good overview of ligation theory is presented in Sambrook et al. pages 1.63-1.67.  Essentially there are two types of reactions that one wants to achieve;  namely limited concatermerisation and There are two types of reactions that one wants to achieve:

1.	Intramolecular ligation.

Recircularization reactions for the formation of deletion plasmids.  In these cases about 1 pmol DNA/ml reaction volume or less should be used.

2.	Intermolecular ligation.

Limited polymerisation between distinct DNA fragments followed by circle closure.

a.	The total DNA concentration should be in the order of 5  10 pmol/ml (vector plus insert) reaction volume.

Use 2X more DNA when prepared by gel elution.

b.	Ratios of vector to insert should be in the order of 1:1 to 1:4 pmol or pmol ends.  If the vector is dephosphorylated, perform the ligation using an excess of vector DNA; ie. a vector to insert ratio of 4:1 pmol.


Plasmid Bluescript pSK
  Plasmid: pSK Bluescript

Contributor:

Babb, B

Plasmid name :

Bluescript

Genbank accession number :

52325 (SK+)

Size (kb) :

2.958kb

Notes :

Plasmid pSK or pKS for single strand DNA production m13 phage etc

map :

Polylinker

    SacI (-)-SacII (?)-BstXI (-)-XmaIII (?)-NotI (+)-XbaI (+)-SpeI (?)-BamHI (+)-SmaI (+)-

                   -PstI (-)-EcoRI (+)-EcoRV (+)-HindIII (+)-ClaI (+)-SalI (+)-XhoI (+)-ApaI (-)-DraII (+)-KpnI (-)

where	(-) is protected from ExoIII nuclease

(+) is susceptible to ExoIII

(?) not checked yet

Unique cloning sites in MCS:

          E

          c                            H

          o               H            i

          O               i            n       E   E

       K  1  A    X     SAn       C    d       c   c

       p  0  p    h     acc       l    I       o   o

       n  9  a    o     lcI       a    I       R   R

       I  I  I    I     III       I    I       V   I

TTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTG

+---------+---------+---------+---------+---------+---------   -->

AACCCATGGCCCGGGGGGGAGCTCCAGCTGCCATAGCTATTCGAACTATAGCTTAAGGAC

650       660       670       680       690       700

                                                  SB

                   B     S     X      EN       D  as       S

           P   S   a     p     b      ao       s  ct       a

           s   m   m     e     a      gt       a  IX       c

           t   a   H     I     I      II       I  II       I

           I   I   I                   /

          CAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGT

     -->  +---------+---------+---------+---------+---------+---------+

          GTCGGGCCCCCTAGGTGATCAAGATCTCGCCGGCGGTGGCGCCACCTCGAGGTCGAAAACA

          710       720       730       740       750       760       770

Enzymes that do not cut:

    AatII    AflII     AgeI    ApaBI     AscI    AvrII     BaeI     BbsI

     BclI    BglII   Bpu10I Bpu1102I    BsaBI    BseRI     BsgI    BsiWI

     BsmI    BsmFI Bsp1407I    BspEI    BspGI    BspMI   BssHII Bst1107I

   BstEII   Bsu36I Eco47III  Eco105I    EcoNI    Esp3I     FseI     HpaI

     MluI     MscI     MunI     NarI     NcoI     NdeI     NheI     NruI

     NsiI     NspV     PacI    PflMI     PmeI     PmlI    PshAI   Psp5II

    RleAI    RsrII    SexAI     SfiI    SgrAI     SphI     SrfI Sse8387I

     StuI     StyI     SwaI  Tth111I     XcmI


Plasmid pUC19
  Plasmid: pUC19

Contributor:

Babb, B

Plasmid name :

pUC19

Accession number :

VB0026

Size (kb) :

2.6

Notes :

map :

Polylinker

   HindIII (+)-SphI (-)-PstI (-)-SalI (+)-XbaI (+)-BamHI (+)-SmaI (+)-KpnI (-)-SacI (-)-EcoRI (+)

where	(-) is protected from ExoIII nuclease

(+) is susceptible to ExoIII

(?) not checked yet

Unique sites in MCS:

                     S

                     s

    H                e  H

    i                8  i         B                  B  E

    n    B    S     P3SAn   X     a    A S     K     aSAc

    d    s    p     s8acc   b     m    v m     p     napo

    I    p    h     t7lcI   a     H    a a     n     IcoR

    I    M    I     IIIII   I     I    I I     I     IIII

    I    I           /                                / /

 GCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCA

 +---------+---------+---------+---------+---------+---------+

 CGGTTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATGGCTCGAGCTTAAGT

 230       240       250       260       270       280       290

Enzymes that do not cut:

    AflII     AgeI     ApaI     AscI    AvrII     BaeI     BbsI     BclI

    BglII   Bpu10I Bpu1102I    BsaAI    BsaBI    BseRI     BsgI    BsiWI

     BsmI    BsmFI Bsp1407I    BspEI    BspGI   BssHII Bst1107I   BstEII

    BstXI   Bsu36I     ClaI   DraIII    DrdII     DsaI     EagI Eco47III

  Eco105I    EcoNI    EcoRV     FseI     HpaI     MluI     MscI     MunI

     NcoI   NgoAIV     NheI     NotI     NruI     NsiI     NspV     PacI

    PflMI     PmeI     PmlI    PshAI   Psp5II    RleAI    RsrII    SacII

     SapI    SexAI     SfiI    SgrAI     SpeI     SrfI     StuI     StyI

     SwaI  Tth111I     XcmI     XhoI


Plasmid pBR322
  Plasmid: pBR322

Contributor:

Babb, B

Plasmid name :

pBR322

Accession number :

?

Size (kb) :

4.363

Notes :

not complete

map :

Polylinker

   none

Unique sites in MCS:

   n/a

Enzymes that do not cut:

????


Competent cells, CaCl2 method, E. coli, long protocol
  CaCl2 competent cells

Description        Short protocol       Related

Contributor:

Zappe, H

Reference:

Dagert and Ehrlich (1979) Gene 6:23-28.

1.	Grow 5 ml overnight of the strain in 2 X YT in a standard container.  Screw cap tight and lie flat on the shaker for good aeration.

2.	Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).

3.	Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)

4.	Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 °C

5.	Keep the cells ice cold in all further steps.

6.	Resuspend the cells in 1/2 culture volume of 0.1 M ice-cold CaCl2.  Hold on ice for minimum of 30 min, prefably 1 - 2 h.

7.	Collect cells as before and gently resuspend them in 1/10 culture volume 0.1 M CaCl2.

8.	Competent cells can be stored almost indefinitely by adding ice-cold sterile glycerol to a final concentration of 10% (v/v).  Mix and leave on ice for 30 min, then store at -70°C

Transformation

1.	To transform, mix 0.1 ml aliquots of cells with DNA (1 - 10 ng).  Leave on ice for 10 - 30 min.

2.	Heat shock at 42°C for 2 min (90 - 120 s is OK)

3.	Add 0.9 ml 2 X YT or LB and allow expression for 30 - 60 min before plating.

4.	If using X-gal and IPTG (optional), place 0.1 ml expression mix on the plate, add 40 µl X-gal and 4 µl IPTG, and spread together.  This ensures that wherever cells go, X-gal does too!.  Better still, add IPTG and X-gal to the plate (0.5 ml X-gal and 50μl IPTG per 100 ml agar).

Media, buffers and solutions

YT,2x

LB

CaCl2 0.1M

MgCl2, 0.1M

ice

glycerol


Competent cells, CaCl2 method, E. coli, short protocol
  CaCl2 competent cells

Description        Long protocol        Related

Contributor:

Zappe, H

Reference:

Dagert and Ehrlich (1979) Gene 6:23-28.

1.	Grow 5 ml E. coli in 2 X YT

2.	Dilute 1ml culture in 100 ml fresh 2 X YT

3.	Grow to OD600 = 0.2 - 0.4

4.	Centrifugation at 5000 rpm for 5 min at 4°C

5.	Resuspend the cells in 50ml of 0.1 M ice-cold MgCl2.

6.	Hold on ice for 30 min

7.	Centrifugation at 5000 rpm for 5 min at 4°C

7.	Resuspend in 10ml of ice cold 0.1 M CaCl2.

8.	Mix and leave on ice for 30 min, then store/use

Transformation

1.	To transform, mix 0.1 ml aliquots of cells with DNA (1 - 10 ng).  Leave on ice for 10 - 30 min.

2.	Heat shock at 42°C for 2 min (90 - 120 s is OK)

3.	Add 0.9 ml 2 X YT or LB and allow expression for 30 - 60 min before plating.

4.	If using X-gal and IPTG (optional), place 0.1 ml expression mix on the plate, add 40 µl X-gal and 4 µl IPTG, and spread together.  This ensures that wherever cells go, X-gal does too!.  Better still, add IPTG and X-gal to the plate (0.5 ml X-gal and 50 μl IPTG per 100 ml agar).

Media, buffers and solutions	Form

YT, 2x

LB

CaCl2 0.1M

MgCl2, 0.1M

ice

glycerol


Competent cells, calcium chloride method, E. coli, description
 CaCl2 Competent Cells

Short protocol        Long protocol        Related

Description:

Competent cells are able to take up DNA....


Transformation of E. coli, calcium chloride method, protocol
  Transformation (CaCl2)

Description        Related

Contributor:

Zappe, H

Reference:

-

1.	To transform, mix 0.1 ml aliquots of cells with DNA (1 - 10 ng).  Leave on ice for 10 - 30 min.

2.	Heat shock at 42°C for 2 min (90 - 120 s is OK)

3.	Add 0.9 ml 2 X YT or LB and allow expression for 30 - 60 min before plating.

4.	If using X-gal and IPTG (optional), place 0.1 ml expression mix on the plate, add 40 µl X-gal and 4 µl IPTG, and spread together.  This ensures that wherever cells go, X-gal does too!.  Better still, add IPTG and X-gal to the plate (0.5 ml X-gal and 50 μl IPTG per 100 ml agar).

Media, buffers and solutions

YT, 2x

LB

IPTG

X-Gal

ice


Transformation of E. coli, calcium chloride method, description
  Transformation (CaCl2 competent cells)

Protocol        Related

Description:

-


Competent cells, E. coli, DMSO method, long protocol
  DMSO competent cells

Description        Short protocol       Related

Contributor:

Zappe, H

Reference:

Chung and Miller (1988) N.A.R. 16:3580.

1.	Grow 5 ml overnight of the strain in 2 X YT in a standard container.  Screw cap tight and lie flat on the shaker for good aeration.

2.	Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).

3.	Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)

4.	Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 °C.

5.	Cells are resuspended in 1/10 th culture volume of ice cold TSB and held on ice for 10 min before transformation.

Transformation

6.	For transformation, 0.1 ml cells are mixed with up to 0.1 µg DNA and held on ice for 10 min.

7.	Add 0.9 ml TSBG and incubate 37 °C for 30 - 60 min before plating.

8.	Competent cells can be frozen in dry-ice/ethanol and stored at -70 °C.

Media, buffers and solutions	Form

YT, 2x

TSB, TSBG

dry/ice ethanol


Competent cells, E. coli, DMSO method, short protocol
  DMSO competent cells

Description        Long protocol       Related

Contributor:

Zappe, H

Reference:

Chung and Miller (1988) N.A.R. 16:3580.

1.	5 ml overnight E. coli in 2 X YT

2.	Dilute overnight culture 1/100 - 1/200 to 25, 50 or 100 ml 2 X YT broth in flasks 5 - 10 X culture volume (i.e. 25 ml in 250 ml flask etc.).

3.	Grow to early log phase (OD600 = 0.2 - 0.4) (90 - 180 min depending on the strain)

4.	Collect cells by centrifugation (4000-5000 rpm for 5 min or 5000 x g for 5 min) at 4 °C.

5.	Cells are resuspended in 1/10 th culture volume of ice cold TSB and held on ice for 10 min before transformation.

Competent cells can be frozen in dry-ice/ethanol and stored at -70°C.

Transformation

6.	For transformation, 0.1 ml cells are mixed with up to 0.1 µg DNA and held on ice for 10 min.

7.	Add 0.9 ml TSBG and incubate 37 °C for 30 - 60 min before plating.

Media, buffers and solutions	Form

YT, 2x

TSB, TSBG

dry/ice ethanol


Competent cells, E. coli, DMSO method, description
 DMSO Competent Cells

Short protocol        Long protocol        Related

Description:

Competent cells are able to take up DNA....


Transformation of E. coli, DMSO method, protocol
  Transformation (DMSO)

Description        Related

Contributor:

Zappe, H

Reference:

-

1.	For transformation, 0.1 ml cells are mixed with up to 0.1 µg DNA and held on ice for 10 min.

2.	Add 0.9 ml TSBG and incubate 37 °C for 30 - 60 min before plating.

Media, buffers and solutions	Form

TSBG

dry/ice ethanol


Transformation of E. coli, DMSO method, description
 Transformation (DMSO competent cells)

Protocol        Related

Description:

Transform DNA into E. coli cells


Plasmid prep: adaptation of Ish-Horowicz method, description
  Plasmid prep: Ish-Horowicz adaption

Short mini-prep protocol        Long protocol       Related

Description:

Mini- midi- and Maxi- plasmid preparation

(Based on Ish-Horowicz and Burke. N.A.R. 9:2989-2998 (1981), Birnboim and Doly N.A.R. 7:1513-1523 (19??), BRL Labs with modifications for CsCl spin.  The method is similar to that described starting on page 1.38, Sambrook et al., with modifications).  Refer to Chapter 1, page 1.21 of Sambrook et al. for further reading.


Plasmid prep: adaptation of Ish-Horowicz method, long protocol
  Plasmid prep: Ish-Horowicz adaption

Short protocol        Related

Contributor:

Zappe, H

Reference:

Based on Ish-Horowicz and Burke. N.A.R. 9:2989-2998 (1981), Birnboim and Doly N.A.R. 7:1513-1523 (19??), BRL Labs with modifications for CsCl spin.  The method is similar to that described starting on page 1.38, Sambrook et al., with modifications).  Refer to Chapter 1, page 1.21 of Sambrook et al. for further reading.

1.	From a single colony on a Luria agar plate, inoculate a 5 ml Luria broth (LB) starter culture with antibiotic selection and incubate to late log (OD600 = 0.6 - 0.8, 6 - 8 h).  Dilute the starter culture 1/100 to the main culture (see note) and shake O/N (16 h).  Ampicillin selection at 100 μg /ml.

Prep type:         miniprep          midiprep        maxiprep

Culture vol.        1.5-4 ml             50 ml            400 ml

Use tube           microfuge      J21 - 50 ml        500 ml

Note:	For maxipreps, usually 200 ml of Luria broth in a 1 liter flask will yield plenty of plasmid (e.g high copy number plasmids such as pUC or Bluescript) but for lower copy number plasmids like pBR322, larger culture volumes of a richer medium may be necessary and/or the plasmids can be amplified by the addition of 170 - 200 μg/ml of Chloramphenicol to the main culture when to OD600 reaches about 0.5.  Continue to shake to culture for a further 12 - 16 h before harvesting (see chapter 1, Sambrook et al.)

2.	Harvest the cells by centrifugation in appropriate rotor (sufficient speed and time to pellet the cells).  Pour off the spent broth to the culture flask, invert the tubes on paper towel to remove the last of the broth and then resuspend completely in the indicated volume of Solution I:

           miniprep        midiprep        maxiprep

Add        0.2 ml            4.0 ml            32 ml

3.	Add solution 2, mix well by shaking and leave on ice for 5 min. (Culture should clear and go gloopy).  This is a timed denaturation step - do not exceed 5 min.

Add        0.4 ml            8.0 ml            64 ml

4.	Add pre-cooled solution 3, shake well and leave at least 5 min on ice:

Add        0.3 ml            6.0 ml            48 ml

Note:	A white flocculate forms - like curds and whey - cellular protein and membrane precipitate.

5.	Spin 10 min (12 K), recover indicated supernatant and transfer to fresh tubes of the same volume.  Avoid contamination with pelleted material (Larger volume preps. can be filtered through cheesecloth):

Recover:    0.9 ml            18.0 ml        144 ml

6.	Add 0.6 ml volumes of isopropanol, mix, leave for 10 min, then spin for 5 - 10 min (12 K for J-21, 8K for GSA).

Add        0.6 ml            10.0 ml        86 ml

7.	Discard the supernatant, wash the pellet in 80 % ethanol, drain thoroughly (i.e. pour off the ethanol, spin the tube briefly and then remove the last drops with a pipette), and resuspend the pellet in TE:

Add        0.3 ml*        1.0 ml            7.5 ml

*	If the miniprep is just going to be used for checking for an insert by restriction, reduce this volume to 50 µl, and use 1 µl in a digest.

The method for maxipreps can now be continued in one of a number of ways: CsCl gradient centrifugation, RNase treatment and phenol extraction (both given below), selective precipitation with polyethylene glycol (page 1.40, Sambrook et al.), or using Nucleobond® column purification.

Media, buffers and solutions	Form

YT

LB

ice

Solution I, II and III

isopropanol

ethanol


Plasmid prep: adaptation of Ish-Horowicz method, CsCl purification
  CsCl gradient centrifugation for Maxipreps.

Long protocol      Plasmid preps       DNA purification

Contributor:

Zappe, H

Note:    Refer to Sambrook et al. for safety regarding the use of Ethidium Bromide.

1.	Add 8.3 g CsCl to the 7.5 ml DNA solution, dissolve and then add 0.8 ml 10 mg/ml EtBr.  Mix and then centrifuge the solution at 10 k/10 min.  Recover supernatant in a fresh tube or standard container.  Adjust Refractive Index to 1.390 and then fill two Vti65 tubes and centrifuge at 50 000 O/N (16 h) at 20 °C.

2.	With visualization under UV (310nm), you should see 1 major band (plasmid) and possibly a minor band above it (residual chromosomal and nicked plasmid DNA).  WEARING GLOVES, first cut off the top of the tube, otherwise, on piercing, air bubbles rush in from the bottom and disrupt the band!!!.  Collect the plasmid band by piercing the bottom of the tube with a needle (20G).  Allow the excess below the band to drip out to a waste container, then collect just the band itself in an eppendorf (control the flow as you would with a pipette).  Discard the remainder in the waste container.  An alternative method for recovering bands is given on page 1.45 of Sambrook et al.

3.	Extract with salt saturated isopropanol (3X) to remove the EtBr (during extraction there are two phases: the EtBr goes to the top (isopropanol) phase and is discarded after each extraction.

4.	Add 2 volumes of water, mix.

5.	Add 1 total volume (new vol.) isopropanol (NOT SALT-SATURATED!), mix, leave 10 min, spin 15 min.  Wash pellet in 70% ethanol and resuspend in TE - 0.5ml.  Determine DNA conc. by 310 - 220 scan.  With pUC plasmids, dilute the DNA 1/50 - 1/100 in TE before scanning.  Peak at about 260. [DNA] = A260 X 50 X dilution. (the factor of 50 is used for dsDNA, 40 for ssDNA and RNA)

Media, buffers and solutions

YT, 2x


Plasmid prep: adaptation of Ish-Horowicz method, RNAse and phenol treatment
  RNase treatment and phenol purification for mini, midi, and maxi preps.

Related        Long protocol      Plasmid preps

Contributor:

Zappe, H

1.	Continue with the volumes as in step 7 of the maxiprep protocol, except make maxiprep to 9 ml TE, and add the appropriate vol. of RNase solution (1 mg/ml heat treated RNase A; 4000 U/ml RNase T1) and incubate at 37 °C for 15 min.

Add        20  μl            0.2 ml            0.5 ml

2.	Add phenol/CHCl3/8-hydroxyquinoline mixture, mix, spin 12 K for 10 min, recover aqueous phase to clean tube.

Add        0.2 ml            0.2 ml            4.0 ml

3.	Add 0.5 volumes of cold 7.5 M NH4-acetate, mix and leave on ice for 15 min.  Spin 12 K for 10 min, transfer supernatant to fresh tube, discard pellet.

Add        0.15 ml        0.6 ml            4.5 ml

4.	Add 0.6 volumes of isopropanol, leave on ice for 10 min, spin 12 K for 15 - 20 min.

Add        0.3 ml            1.1 ml            8.5 ml

5.	Wash pellet with cold 80% ETOH, drain properly or spin briefly and remove last drops of ETOH.

6.	Dissolve DNA in TE.

Add        50 μl            0.2 ml            0.5 ml

7.	Store in aliquots at -20 °C or better at -70 °C

Media, buffers and solutions

YT, 2x


Plasmid prep: adaptation of Ish-Horowicz method, mini-prep notes
  Mini-prep Notes - no phenol extraction

related

Contributor:

Zappe, H

1.	An important point in getting the DNA to restrict, directly after resuspending in TE, is the ratio of miniprep DNA to final digestion vol. i.e. dilute the prep at least 10 X in the digest, preferably 20 X (1 μl in 20 μl digest).  If it does not restrict then a second ETOH ppt may help.  Otherwise the prep. may need phenol extraction.

2.	Culture volumes may vary from 1 to 5 ml, but this prep gives good yields, 2 μl being enough to see on a gel.

3.	Include RNAse in the tracking dye (200 μg/ml).

4.	The cells can be left for some hours in solution 1.  DNA pellets can be left in 70% ETOH.

5.	The amounts of solutions 1, 2 and 3 can vary but keep the ratios the same (1:2:1.5).


Plasmid prep: adaptation of Ish-Horowicz method, short protocol - miniprep
  Mini-prep method - short protocol

related        Long protocol

Contributor:

Zappe, H

1.	Grow a 5 ml O/N culture with antibiotic selection

2.	Collect cells from 4 ml culture in a 2 ml Eppendorf tube.

3.	Resuspend cell pellet properly in 0.2 ml solution 1.

4.	Leave at room temperature for minimum 5 min.

5.	Add 0.4 ml solution 2, mix, 5 min on ice.

6.	Add 0.3 ml pre-cooled solution 3, mix, 5 min on ice.

7.	Spin 5 min

8.	Spin 5 min and recover 0.9 ml supernatant.  Add 0.6 ml isopropanol, mix, leave for 2 min , spin for 5-10 min.

9.	Retain pellet and wash with 0.5 ml 70% ETOH.  Pour off of the ETOH, dab dry, spin and remove remainder by pipet.

10.	Air dry for 5 min, resuspend pellet in 50 - 100 μl TE.  Allow to resuspend for about 10 min at RT.  Store at 4 degC.

Media, buffers and solutions


Plasmid prep: JAT-preps, description
  Plasmid prep: JAT prep

protocol Related

Description:

I received the method from Helen Stutz and I believe it to originate from Prof JA Thomson.  There is no reference available for the method.  It is a very quick way to test whether a E. Coli colony contains a plasmid with an insert.  The DNA is electrophorezed without digestion and ccc (closed-circular covalent) DNA of comparitive size is run as a control.


Plasmid prep: JAT prep,  protocol
  Plasmid prep: JAT preps, protocol

Description        Related

Contributor:

Stutz, H

Reference:

?

1.	Toothpick colonies into 0.6ml LB with antibiotic selection.

2.	Incubate with haking for more than 4 hours.

3.	Spin eppendorfs at maximum speed for 30sec

4.	Resuspend the pellet in 60 μl STE

5.	Add 60 μl of phenol-chloroform-isoamyl alcohol (25:24:1) mix

6.	Vortex for 15 sec

7.	Spin for 5 min

8.	Remove supernatant to a fresh tube

9.	Load 10 μl for gel electrophoresis (run relative to uncut plasmids of known size).

10.	Constructs of potentially the correct size can be preicipitated by normal methods for further analysis.

Media, buffers and solutions	Form

STE


DNA restriction enzyme endonuclease digestion, description
  DNA restriction endonuclease digestion

Related        Long protocol        λ DNA standards

Restriction enzymes are used to cut DNA at specific DNA sequences....

See also restriction enzyme sites


DNA restriction enzyme endonuclease digestion, long protocol
  DNA restriction endonuclease digestion

Related       Descriptions        λ DNA standards        R/E sites

Contributor:

Zappe, H

Reference:

1.	Restriction digests are carried generally using the specific buffer or buffer system provided by the manufacturer.  The enzyme SmaI requires a unique buffer .

2.	Multiple restrictions, requiring different enzymes, can be combined in a single digest provided the buffers are compatible.

3.	If this is not possible, then the digestion is done sequentially using the enzyme with the lowest salt and highest temperature requirement first and the salt concentrations and incubation temperatures adjusted before the addition of the next enzyme.

4.	The incubation temperature is 37 °C for all enzymes with a few exceptions.  eg. BclI and TaqI (65°C).

5.	Digestion volumes vary depending on the amount of DNA being digested but normally 200-300 ng DNA is digested in a 20 μl volume using 1 or more units of enzyme for 1 h.  For digestions of plasmids where only a few fragment are expected, 50 - 100 ng would be enough to visualise on an agarose gel.

7.	Miniprep DNA is always prepared sufficiently concentrated so that only 1 μl  need be digested in a 20 μl  reaction.  This dilution is important as impurities could inhibit restriction at higher concentrations.

8.	 Concentrated enzyme stocks are diluted to 1 or 2 units/ μl  using a universal restriction enzyme dilution buffer (below).  Opinions differ as to the necessity of this step, and with the advent of adjustible 2 μl pipettes, very small volumes of the concentrated enzyme stock can be dispenced.

9.	For electrophoretic analysis, digestions are stopped by the addition of DNA sample loading solution.  If a sample is to be used for further enzyme reactions (eg. ligation), purify by the Clean-up method or geneclean.

Media, buffers and solutions

Restriction enzyme dilution buffer

Restriction enzyme core reaction buffers

Sma I reaction buffer


DNA digestion: restriction enzyme dilution buffer
  Restriction enzyme dilution buffer

Contributor:

Zappe, H

Related:

DNA digestion

Storage:

-20°C

Restriction enzyme dilution buffer

Component 	stock conc	final conc.	/ 10ml

Tris-HCl, pH 7.5	1M	10mM	0.1ml

NaCl	5M	50mM	0.1ml

β mercapto-ethanol	10mM	7 μl

Gelatin	10mg/ml	100 μg/ml	0.1ml

water	5.3ml

glycerol	44%(v/v)	4.4 ml


DNA digestion: restriction enzyme core reaction buffer
  Restriction enzyme core buffers (10X).

Contributor:

Zappe, H

Related:

DNA digestion

Storage:

-20°C

Restriction enzyme dilution buffer (10x)

Salt concentration mM

Component 	stock conc	0	50	100	150

Tris-HCl, pH 7.5	1M	1ml	1ml	1ml	1ml

NaCl	5M	0	1ml	2ml	87.7mg

BSA (albumin)	10mg/ml	1ml	1ml	1ml	1ml

MgCl2	1M	1ml	1ml	1ml	1ml

DTT	0.5M	0.2ml	0.2ml	0.2ml	0.2ml

water	2.4ml	1.4ml	0.4ml	2.4ml

glycerol	44%(v/v)	4.4 ml	4.4 ml	4.4ml


DNA digestion: Sma I restriction enzyme reaction buffer
  Sma I restriction enzyme reaction buffer

Contributor:

Zappe, H

Related:

DNA digestion

Storage:

-20°C

Sma I restriction enzyme reaction buffer (10x)

Component 	stock conc	final conc.	/ 10ml

Tris-HCl, pH 8.0	1M	0.1M	1ml

KCl	5M	0.2M	2ml

MgCl2	1M	0.2M	2ml

DTT	0.5M	0.2ml	0.2ml

water	0.4ml

glycerol	44%(v/v)	4.4ml

All the components, except the glycerol, are combined in a 10 ml sterilin tube.  The tube is then filled to the 10 ml mark with sterile glycerol.  Mix by gently shaking the tube.  The volume marks on the sterilin tube are used as it is not possible to accurately pipette 5 ml of undiluted glycerol.


Recipe for TSB and TSBG from DMSO competent cells
  TSB and TSBG

Contributor:

Zappe, H

Related:

DMSO transformation

TSB

Component 	stock conc	final conc.	/ 100 ml	/1l	/10ml

Peptone	-	1.6 g

Yeast extract	-	1.0 g

NaCl	-	0.5 g

PEG (mw 3350-4000)	10 % (w/v)	10.0 g

DMSO	5 % (v/v)	5.0 ml

MgCl2	(1 M)	10 mM	1.0 ml

MgSO4	(1 M)	10 mM	1.0 ml

Water	-	to 100 ml

This solution can also be made using LB.  If ready made broth is available, one can simply add the PEG, DMSO and Mg salts to it - the level of dilution is not significant.

TSBG

TSB plus 20 mM glucose (2 ml 20% (w/v) glucose/100 ml TSB


Recipe for DNA extraction cell suspension buffer
  Cell suspension buffer

Contributor:

Zappe, H

Related:

Bacterial DNA extraction

Cell suspension buffer:

Component 	stock conc	final conc.	/ 100 ml	/1l	/10ml

Tris-HCl (pH 8.0)	1M	10mM	1ml

EDTA (pH 8.0)	0.5M	1mM	0.2ml

sucrose	-	0.35M 	12g

water	to 100ml

If the cells are going to be physically disrupted (French press or glass beads), then do not add the sucrose.


Recipe for DNA extraction lysing solution
  Lysing solution

Contributor:

Zappe, H

Related:

Bacterial DNA extraction

Lysing solution  (2X):

Component 	stock conc	final conc.	/ 100 ml	/1l	/10ml

Tris-HCl (pH 8.0)	1M	100mM	10ml

EDTA (pH 8.0)	0.5M	20mM	4ml

NaCl	5M	0.3M	6ml

SDS	25%(w/v)	2%	8ml

β-mercaptoethanol	2%(v/v)	2ml

proteinase K	20mg/ml	100μg/ml	0.5ml

water	to 100ml

Add proteinase K (50 l of a 20 mg/ml stock solution per 10 ml 2X lysing solution) and the -mercaptoethanol to the amount of lysing solution needed.  The other components can be premixed as a stock solution.


Recipe for sodium perchlorate
  Sodium perchlorate

Contributor:

Zappe, H

Related:

many protocols

Caution:

can be explosive

sodium perchlorate

Component 	stock conc	final conc.	/ 10 ml	/100ml

NaClO4  	-	5M	7g	70g

Filter sterilize


Recipe for TE Buffer
  TE buffer

Contributor:

Zappe, H

Related:

many protocols

TE Buffer

Component 	stock conc	final conc.	/ 10 ml	/100ml	/1l

Tris, pH 8.0	1M	10mM	0.1ml	1ml	10ml

EDTA, pH 8.0	0.5M	1mM	20μl	0.2ml	2ml ??


Recipe for chloroform:isoamyl alcohol mixture
  Chloroform:isoamyl alcohol mix

Contributor:

Zappe, H

Related:

many protocols

Care:

caution

Chloroform:Isoamyl (24:1)

Component 	stock conc	final conc.	/ 10 ml	/100ml

Chloroform	96ml

Isoamyl alcohol	4ml


Recipe for buffered phenol
  Phenol

Contributor:

Zappe, H

Reference:

Methods in Microbiology 21:141 (1988)

related:

many protocols

storage:

-20°C

Care:

caution

Buffered Phenol (pH 7.6, 10mM Tris)

Component 	stock conc	final conc.	??	/100ml

8-hydroxyquinoline	0.6g

NaOH	2M	7.5ml

Water	130ml

Tris-HCl, pH 7.5	1M	6ml

Phenol	500g

leave overnight to liquefy

aliquot and store at -20°C


Recipe for DNA extraction RNAse mix
  RNAse mix for DNA extraction

Contributor:

Zappe, H

Related:

Bacterial DNA extraction

RNAse mix

Component 	stock conc	final conc.	100μl	/100ml

RNAse A 	10mg/ml	1mg/ml	10μl

RNAse T1	100U/μl	4000U/ml	4μl

TE buffer	86μl


Recipe for ammonium acetate
  Ammonium acetate

Contributor:

Zappe, H

Related:

many protocols

Storage:

4°C

Ammonium acetate

Component 	stock conc	final conc.	/ 200 ml	/1l

Ammonium acetate	-	7.5M	289g

water	to 200ml

Can be used as Solution III for plasmid preparation.


Recipe fo ampicillin
  Ampicillin

Contributor:

Zappe, H

Related:

many protocols

Storage:

-20°C

Ampiciliin (Sigma, sodium salt), 1000x

Component 	stock conc	final conc.	/ 20 ml	/1l

Ampicillin	-	100 mg/ml	2g

water	to 20ml

filter sterilize and store aliquots at -20°C

dilute 1/1000 for final conc of 100 μg/ml


Recipe for ATP
  ATP

Contributor:

Zappe, H

Related:

many protocols

Storage:

-20°C

ATP (0.1M)

Component 	stock conc	final conc.	/ 0.8ml ml	/1l

ATP	0.1M	60mg

NaOH	0.1M	adjust to pH 7.0

water	to 0.8ml

Adjust to pH 7.0 with 0.1M NaOH (spotting a few μl on pH paper)

Freeze 0.1ml aliquots


Recipe for calcium chloride
  Calcium chloride

Contributor:

Zappe, H

Related:

many protocols

Storage:

4°C

Calcium chloride (1M), CaCl2

Component 	stock conc	final conc.	/ 100 ml	/1l

CaCl2.2H20	-	1M	14.7g

water	to 100ml

autoclave


Recipe for DTT
  Dithiothreitol (DTT)

Contributor:

Zappe, H

Related:

many protocols

Storage:

-20°C

Dithiothreitol (DTT)

Component 	stock conc	final conc.	/ 20 ml	/1l

DTT	-	1M	3.09g

sodium acetate, pH 5.2	to 20ml

filter sterilize and store aliquots at -20°C


Recipe for EDTA
  EDTA (ethyl??)

Contributor:

Zappe, H

Related:

many protocols

EDTA (Ethylene??)

Component 	stock conc	final conc.	/ 400 ml	/1l

EDTA	-	0.5M	93.05g

NaOH	+/- 10g

water	to 400ml

EDTA will not dissolve until +/- pH 8.0

autoclave


Recipe for ethidium bromide
  Ethidium bromide (EtBr)

Contributor:

Zappe, H

Related:

many protocols

Care:

 never breath dust - mutagen

Ethidium bromide (EtBr)

Component 	stock conc	final conc.	/ 10 ml	/1l

EtBr	-	10mg/ml	0.1g

water	to 10ml


Recipe for ethanol stock
  Ethanol (EtOH)

Contributor:

Zappe, H

Related:

many protocols

Ethanol (EtOH)

Component 	stock conc	final conc.	/ 500 ml	/1l

EtOH	-	70%	350ml

water	150ml


Recipe for glucose stock solution
  Glucose stock solution

Contributor:

Zappe, H

Related:

many protocols

Glucose

Component 	stock conc	final conc.	/ 100 ml	/1l

Glucose	-	20%(w/v)	20g

water	to 100ml

autoclave


Recipe for phenol-chloroform-isoamyl alcohol mix
  Phenol Chloroform mix

Contributor:

Zappe, H

Related:

many protocols

Care:

Storage:

-20°C

Phenol-Chloroform-Isoamyl Alcohol

Component 	stock conc	final conc.	/ 20 ml	/1l

Phenol	-	50%	10ml

Chloroform:isoamyl alcohol	50%	10ml

alternate recipe:

Phenol-chlorofrom-isoamyl alcohol

Component 	stock conc	final conc.

Phenol	-	50%	500g

water	until 2 phases

8-hydroxy quinoline	0.1%(w/v)	?

Chloroform:isoamyl alcohol	50%	1 volume

2.	Melt the phenol in a 50°C water bath, and then add dH2O until the solution is saturated (2 phases visible).  Add 0.1% w/v 8-hydroxy quinoline.  Then  mix 1:1 with the chloroform-isoamyl solution (pers. comm. J.L. Johnson, Virginia Tech.).


Recipe for IPTG
  IPTG

Contributor:

Zappe, H

Related:

many protocols

Storage:

-20°C

IPTG. (Isopropyl-ß-D-thio-galactopyranoside).

Component 	stock conc	final conc.	/ 10 ml	/1l

IPTG	-	200mg/ml	2g

water	to 10ml

filter sterilize and store aliquots at -20°C


Recipe for salt-saturated isopropanol
  Isopropanol, salt-saturated

Contributor:

Zappe, H

Related:

many protocols

Salt-saturated isopropanol

Component 	stock conc	final conc.	/ 400 ml	/1l

isopropanol	-	-	300ml

NaCl	5M	100ml

A white precipitate forms and two phases appear.  Use the top phase.


Recipe for 1M magnesium chloride
  Magnesium chloride

Contributor:

Zappe, H

Related:

many protocols

Magnesium chloride, MgCl2 (1M)

Component 	stock conc	final conc.	/ 100 ml	/1l

MgCl2.6H2O	-	1M	20.3g

water	to 100ml

Autoclave.


Recipe for magnesium sulphate
  Magnesium sulphate

Contributor:

Zappe, H

Related:

many protocols

Magnesium sulphate, MgSO4 (1M)

Component 	stock conc	final conc.	/ 100 ml	/1l

MgSO4 (.7H20??)	-	1M	24.7g

water	to 100ml

autoclave.


Recipe for 5M potassium acetate
  Potassium acetate

Contributor:

Zappe, H

Related:

many protocols

Storage:

-4°C

Potassium acetate, KAc (5M)

Component 	stock conc	final conc.	/ 100 ml	/1l

KAc	-	5M	49.1g

water	to 100ml

autoclave.

This is the preferred Solution III for plasmid preparation.


Recipe for Sodium dodecyl sulphate
  Sodium dodecyl sulphate

Contributor:

Zappe, H

Related:

many protocols

Care:

sodium dodecyl sulphate, laural sulphate (SDS) (25%)

Component 	stock conc	final conc.	/ 100 ml	/1l

SDS	-	25%(w/v)

water	to 100ml

heat to about 80°C to dissolve and add a few fdrops of hydrochloric acid to pH 7.2


Recipe for sodium acetate
  Sodium acetate

Contributor:

Zappe, H

Related:

many protocols

sodium acetate, NaAc (pH 5.2 or 6.0)

Component 	stock conc	final conc.	/ 500 ml	/100ml

sodium acetate	-	3M	204.5g

water 	to 400ml

glacial acetic acid	to required pH

water (after pH adjust)	to 500ml

make to pH 5.2 or to pH 6.0

an alternative solution III for plasmid preparation.


Recipe for sodium chloride
  Sodium chloride

Contributor:

Zappe, H

Related:

many protocols

Sodoium chloride, NaCl (5M)

Component 	stock conc	final conc.	/ 100 ml	/1l

NaCl	-	5M	29.22g

water	to 100ml

autoclave.


Recipe for sodium hydroxide (10M)
  Sodium hydroxide

Contributor:

Zappe, H

Related:

many protocols

Care:

caustic

Sodium hydroxide, NaOH, (10M)

Component 	stock conc	final conc.	/ 100 ml	/1l

NaOH	-	10M	40g

water	to 100ml

store in a plastic container

dilute to make stocks of 0.1M and 1M


  Magnesium sulphate

Contributor:

Zappe, H

Related:

many protocols

Magnesium sulphate, MgSO4 (1M)

Component 	stock conc	final conc.	/ 100 ml	/1l

MgSO4 (.7H20??)	-	1M	24.7g

water	to 100ml

autoclave.


Recipe for Tris-acetate-EDTA buffer
  Tris-acetate-EDTA buffer

Contributor:

Zappe, H

Related:

agarose gel electrophoresis

Tris-acetate-EDTA buffer, TAE (50x)

Component 	stock conc	final conc.	/ 1l

Tris-HCl	-	?M	242g

Glacial acetic acid	?	57.1ml

EDTA	0.5M	?	100ml

water	to 1000ml

dilute 200ml in 10l water for 1x


Recipe for Tris buffer
  Tris Buffer (1M)

Contributor:

Zappe, H

Related:

many protocols

Tris (trihy...)

Component 	stock conc	final conc.	/ 100 ml	/1l

TRIS	-	1M	12.1g

water	to 80ml

concentrated HCl	adjust to required pH

water	to 100ml

Three solutions are generally used:

pH 7.4  -  add about 7.0 ml HCl

pH 7.6  -  add about 6.0 ml HCl

pH 8.0  -  add about 4.2 ml HCl


Recipe for tRNA
  tRNA

Contributor:

Zappe, H

Related:

DNA protocols

Storage:

4°C

tRNA (1mg/ml)

Component 	stock conc	final conc.	/ 10 ml	/1l

tRNA (E. coli)	-	1mg/ml	10mg

water	to 10ml


Recipe for X-gal
  X-Gal

Contributor:

Zappe, H

Related:

clone detection

Storage:

-20°C

Reference:

Biotechniques 15(2):292, August, 1993).

Notes :

light sensitive

5-bromo-4-chloro-3-indolyl-ß-D-galactoside, X-Gal (20mg/ml)

Component 	stock conc	final conc.	/ 10 ml

X-gal	-	20mg/ml	200mg

DMSO	to 5ml

once dissolved, water	to 10ml

Add 0.5 ml X-gal solution to 100 ml agar.


Recipe for yeast tryptone medium
  Yeast tryptone medium

Contributor:

Zappe, H

Related:

E. coli growth

Yeast tryptone broth/agar, 2xYT

Component 	stock conc	final conc.	/1l

Tryptone	16g

Yeast extract	10g

NaCl	5g

for agar plates

agar	15g

autoclave.


Recipe for terrific broth
  Terrific broth

Contributor:

Zappe, H

Related:

E. coli growth

Reference:

BRL Focus 9(2):12, 1987.

Terrific Broth

Component 	stock conc	final conc.	/1l

Bacto-tryptone	12g

Bacto-yeast extract	24g

glycerol	4ml

water	to 900ml

autoclave, then add

sterile potassium salts	100ml

for higher yields of plasmid: pUC18 should give about 0.9 mg DNA per 100 ml broth


Recipe for potassium salts
  Potassium salts

Contributor:

Zappe, H

Related:

Terrific broth

Potassium salts for Terrific broth

Component 	stock conc	final conc.	/1l

KH2PO4	0.17M

K2HPO4	0.72M

autoclave.


Recipe for solution I, plasmid preps
  Solution I
Plasmid prep

Contributor:

Zappe, H

Related:

Plasmid prep

Plasmid prep solution I (10x)

Component 	stock conc	final conc.	/100ml

Tris-HCl, pH 8.0	1M	25mM	2.5ml

EDTA, pH 8.0	0.5M	50mM	10ml

Glucose	20%(w/v)	1%(w/v)	5ml

water	82.5ml

Note:	do not autoclave this solution once the components have been mixed as the glucose caramelises.


Recipe for solution II, plasmid prep
  Solution II
Plasmid prep

Contributor:

Zappe, H

Related:

Plasmid prep

Storage:

Make up fresh weekly

Plasmid prep solution II (1x)

Component 	stock conc	final conc.	/100ml	/10ml

NaOH	10M	0.2M	2.0ml	.2ml

SDS	25%(w/v)	1%(w/v)	4ml	0.4ml

water	94ml	9.4ml


Recipe for solution III, plasmid prep
  Solution III
Plasmid prep

Contributor:

Zappe, H

Related:

Plasmid prep

Plasmid prep solution III (1x)

Component 	stock conc	final conc.	/100ml	/10ml

Potassium acetate	5M	3M	60ml	6ml

glacial acetic acid	11.5ml	1.15ml

water	28.5ml	2.85ml


Recipe for LB
  Recipes: Luria broth

Contributor:

Zappe, H

Related:

E. coli growth

LB

Component 	stock conc	final conc.	/ 100 ml	/1l	/10ml

Tryptone

NaCl

Yeast extract

Water	-	to 100 ml


Lisy of all references
References

List of papers referred to in this Help file.  See also contributing authors

Sambrook et al., 1991, A laboratory manual.  Press etc

Dagert and Ehrlich (1979) Gene 6:23-28.

Calcium chloride competent cells

Chung and Miller (1988) N.A.R. 16:3580.

DMSO competent cells

Ish-Horowicz and Burke. N.A.R. 9:2989-2998 (1981).

plasmid prep

Birnboim and Doly N.A.R. 7:1513-1523 (19??)

plasmid prep

Marmur (J. Mol. Biol. 3:208-218, 1961).  Modified by J.L. Johnson (Virginia Tech.)

DNA extraction

Methods in Microbiology 21:141 (1988)

phenol preparation

Biotechniques 15(2):292, August, 1993).

X-gal

BRL Focus 9(2):12, 1987.

terrific broth

Struhl, K (1985), BioTechniques 3(6):452-453.)

dna ligation in agarose

H Hansen and H Lemke (1993), Biotechniques 14(1):28-29.

direct band elution during agarose gel electrophoresis.

Smith and Summers, (1980),  Anal. Biochem. 109:123-129

Southern transfer of DNA

Rigby et al., 1977. J. Mol. Biol. 113:237.

Nick - Translation of DNA probes and spin-column purification

Meinkoth and Wahl (1984) (Anal. Biochem. 138:267)

The alkali denaturing method

Towbin et al. (1979). ??

Western blots

(modified from Reed & Mann 1985. NAR 13: 7207-7221)

Southern blots

Modified from Cold Spring Harbour Manual)

spin columns

(Modified from Church & Gilbert 1984. PNAS 81: 1991-1995)

DNA_DNA hybridization

The following refer to PCR methods

---------------------------------------------------

Compton T (1990).  Degenerate primers for DNA amplification.pp. 3945 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Fuqua SAW, Fitzgerald SD and McGuire WL (1990).  A simple polymerase chain reaction method for detection and cloning of lowabundance transcripts.  BioTechniques 9 (2):206211.

Gelfand DH and White TJ (1990).  Thermostable DNA polymerases.  pp. 129141 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Innis MA and Gelfand DH (1990).  Optimization of PCRs.  pp. 312 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Krawetz SA, Pon RT and Dixon GH (1989).  Increased efficiency of the Taq polymerase catalysed polymerase chain reaction.  Nucleic Acids Research 17 (2):819.

Rybicki EP and Hughes FL (1990).  Detection and typing of maize streak virus and other distantly related geminiviruses of  grasses by polymerase chain reaction amplification of a conserved viral sequence.  Journal of General Virology 71:25192526.

Rychlik W, Spencer WJ and Rhoads RE (1990).  Optimization of the annealing temperature for DNA amplification in vitro.  Nucleic Acids Research 18 (21):64096412.

Sarkar G, Kapeiner S and Sommer SS (1990).  Formaqmide can drastically increase the specificity of PCR.  Nucleic Acids Research 18 (24):7465.

Smith KT, Long CM, Bowman B and Manos MM (1990).  Using cosolvents to enhance PCR amplification.  Amplifications 9/90 (5):1617.

Thweatt R, Goldstein S and Reis RJS (1990).  A universal primer mixture for sequence determination at the 3' ends of cDNAs.  Analytical Biochemistry 190:314316.

Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991).  The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.  DNA and Cell Biology 10 (3):233238.

Yap EPH and McGee JO'D (1991).  Short PCR product yields improved by lower denaturation temperatures.  Nucleic Acids Research 19 (7):1713.


All replated topics
    List of related topics

DNA DNA clean-up dephosphorylation DNA ends DNA labelling ligations precipitation purification reactions and restriction recovery shortening DNA transfer  DNA sequencing	Gel electrophoresis of DNA Southern blots  Bacterial Plasmid extractions DNA extractions Transformation  Protein Western blots  Protein gels	PCR  PCR Applications PCR products PCR Miscellaneous Primer design Reaction conditions PCR Sample reaction PCR Sequencing RNA


    Related topics

DNA
DNA clean-up
DNA dephosphorylation
DNA ends
DNA labelling
DNA ligations
DNA precipitation
DNA purification
DNA reactions and restriction
DNA recovery
DNA shortening
DNA transfer
DNA sequencing

Gel electrophoresis of DNA


    Related topics

RNA


    Related topics

Bacterial
Plasmid extractions
DNA extractions
Transformation


    Related topics

Protein
Western blots
Protein gels


    Related topics

Total DNA extraction
DNA extraction description (Zappe)
Genomic DNA prep (Coyne)

Plasmid extraction


    Related topics

Protocols
Long protocol (mini-, midi-, maxi- preps) (Zappe)
Short protocol (minipreps) (Zappe)
Mini-prep notes (Zappe)
JAT preps (Stutz)

Purification

Total DNA extraction


    Related topics

Competent cells

    CaCl2 method

    DMSO method

Transformation

    Heat shock for CaCl2

    DMSO


    Related topics

DNA reactions
DNA shortening
DNA shortening
DNA molecule ends
DNA ligations
DNA digestion

See also
restriction enzyme sites


    Related topics

DNA shortening
Bal31 nuclease
ExoIII (Heinekoff)


    Descriptions of Related topics

DNA ends manipulation for ligation
Fill in
Nibble-back
Partial fill-in


    Related topics

DNA recovery from agarose
description
direct elution
electroelution
Geneclean


    Related topics

DNA electrophoresis
Notes on agarose (Zappe)
Resolution of DNA
λ standards
λ-PstI


    Related topics

DNA ligations
Ligation theory
ligation in agarose gels
amounts of DNA to use
Protocol descriptions
notes on ligation
Hints on troubleshooting

Protocols
with ligation mixes (A)
tube reactions (B)


    Related topics

SDS-PAGE gels (Zappe)
Description
Inclusive protocol
Gels
Electrophoresis
Staining/destaining
Drying

Other SDS-PAGE protocols


    Related topics

Southern blots (Zappe)
Description
Pre-treatment
Nick translate
Spin column purify
Transfer
Wash
Autorads

Other Southern methods


    Related topics

DNA transfer
mono-directional
bi-directional
Gel prep and transfer (Coyne)

See also Southerns


    Related topics

DNA purification
spin column purification (Zappe)
spin column purification (Coyne)

Extra purification
Cesium chloride gradient centrifugation for maxipreps
RNAse treatment and phenol purification


    Related topics

Southern Blots
32P (Zappe) (entire method)
DNA hybridization (modified Church, Coyne)


    Related topics

DNA labelling
Nick translation


    Related topics

SDS-PAGE gels
Zappe protocol


    Related topics

Western blots
Zappe protocol (BM)


    Related topics

Clean-up of DNA reactions
Descriptions
Phenol/Chloroform (Zappe)
Geneclean


    Related topics

DNA precipitation
Ammonium acetate/ethanol (Coyne)
Isopropanol
Lithium chloride


    Related topics

DNA dephosphorylation
Description (Coyne)
Typical reaction (Coyne)


    Related topics

PCR
Applications
Dealing with products
Miscellaneous
Primer design
Reaction conditions
Sample reaction
Sequencing PCR Products


    Related topics

PCR Applications
Asymmetric PCR for ssDNA, description (Rybicki)
cDNA PCR , description (Rybicki)


    Related topics

PCR Reaction conditions
Annealing temperature and time, description (Rybicki)
Cycle number, description (Rybicki)
Denaturing temperature and time, description (Rybicki)
Elongation temperature and time, description (Rybicki)
Helix Destabilisers / Additives, description (Rybicki)
"Hot Start" PCR:, description (Rybicki)
Initial Conditions, description (Rybicki)
Reaction buffer, description (Rybicki)
Reagent Concentrations, description (Rybicki)
Temperature cycling, description (Rybicki)


    Related topics

PCR Primer design
simple rules for primer design, description (Rybicki)
Degenerate Primers, description (Rybicki)
Incorporation of Restriction Sites, description (Rybicki)
Primer Length, description (Rybicki)


    Related topics

PCR Dealing with products
Cleaning PCR Products, protocol (Rybicki)
Detecting Products, description (Rybicki)
Labelling PCR Products with Digoxigenin, description (Rybicki)
T-A Cloning Strategy, description (Rybicki)


    Related topics

PCR Sequencing
Sequencing PCR Products, description (Rybicki)


    Related topics

PCR Miscellaneous
rePCR unique bands (Rybicki)
introduction
protocol
summary notes, (Rybicki)


    Related topics

PCR Sample reaction
Sample reaction


    Related topics

DNA sequencing
Pharmacia
Pharmacia - GC rich
Promega taqtrack deaza sequencing
Extra long gels
Gel preparation
Gel pouring
setting up sequencing apparatus
notes on sequencing techniques

See also
notes on sequencing techniques
PCR based sequencing
Sequencing gel preparation


    Related topics

DNA sequencing  pharmacia protocol
Alkaline denaturation of ds template
Priming reaction
Termination tubes
Labelling reaction
Termination reaction

DNA sequencing  pharmacia gcrich protocol
Alkaline denaturation of ds template
Priming reaction
Termination tubes
Labelling reaction
Termination reaction

See also
notes on sequencing techniques
Pharmacia description
Other sequencing protocols


    Related topics

DNA sequencing  promega taqtrack deaza protocol
Alkali denaturation
Annealing ds plasmid template and primer
Termination tubes
Extension/labelling reaction
Termination reaction

See also
notes on sequencing techniques
Promega description
Other sequencing protocols


    Related topics

Sequencing apparatus
Extra long gels
Gel preparation
Gel pouring
setting up sequencing apparatus

See also
notes on sequencing techniques
Sequencing protocols


SDS-PAGE protein gels, description
  SDS-PAGE gels (Zappe)

Related        Other SDS-PAGE        Protocol (all)

Description:

SDS-Polyacrylamide Gel Electrophoresis of proteins

1.	inclusive protocol

2.	Gel pouring

2.	Electrophoresis

2.	Staining

2.	Drying


Protein, SDS-PAGE gels, long protocol
  SDS-PAGE gels: inclusive protocol

Related

Contributor:

Zappe, H

Reference:

-

Caution:

acrylamide is an accumlative neurotoxin and use a resipirator for TEMED

Pouring the gel

1.	Wash plates and spacers, rinse in distilled water and allow to dry.

2.	Degrease plates by wiping with ethanol, and when dry remove any dust with a fine brush.

3.	Put glass plates together with spacers about 2 mm in from edge.  Clip plates together.

4.	Prepare 25 ml 2% agar and melt.  Seal edges with agarose.

5.	Set up plates vertically.

6.	Make sufficient resolving gel mixture containing everything except the APS and TEMED, and de-gas for 10 min.

7.	Add APS and TEMED to degassed preparation, mix and dispense between plates. Avoid trapping air bubbles.

8.	Overlay with water saturated butanol, and allow to polymerise (10 min to 1 h)

9.	After polymerization of separation gel, pour away butanol and unpolymerized acrylamide, rinse with distilled water and allow to drain.  Use Whatman's No 1 paper to dry the top gel surface as much as possible.

10.	Make sufficient staking gel mixture (without APS and TEMED) and de-gas as before

11.	Add APS and TEMED, mix and dispense into avoiding air bubbles.

12.	Insert comb and allow to polymerize (about 1h).

Preparing the gel for electrophoresis

1.	Remove clips from bottom-end of gel and CAREFULLY remove lower spacer.

2.	Remove comb CAREFULLY, wash wells and lower gel edge with distilled water.

3.	Place running buffer in lower buffer tank.

4.	Place gel into apparatus and secure with clips.

5.	Fill upper buffer tank to about 5mm above the gel plate.

6.	Remove bubbles trapped under gel, using siringe with curved needle.  Take care not to pry plates apart.

7.	Add samples with Hamilton syringe.Ensure EACH well is filled with the SAME volume.

8.	Plug leads into power pack

9.	Run the gel at 100 Volts to stack the proteins.  When the proteins enter the separating gel increase to 200 V (about 20 mA per gel).

Staining

????

Gel drying

1.	Destained gels should be washed AT LEAST 4 times in 10% acetic acid to remove ethanol.

2.	Prevent gel cracking by soaking gel in 10% acetic acid containing 1% glycerol for 45 min prior to drying.

3.	Transfer gel on filter paper to dryer heating block, and remove any air bubbles trapped between the gel and the paper before drying is commenced.

4.	Check the red silicone sheet of the gel dryer for cracks before use.

5.	Cover gel with red silicone sheet and connect the vacuum pump.

6.	Turn on the heating element by turning on the timer.  Dry for about 60 min.

7.	Disconnect vacuum tube from dryer before opening red silicone sealing sheet.

Media, buffers and solutions	Form

tank buffer

sample buffer

resolving buffer
stacking buffer
acrylamide stock
ammonium persulphate
gel mixes
stain
destain
TEMED


SDS-PAGE protein gels, pouring
  SDS-PAGE gels

Related

Contributor:

Zappe, H

-

Caution:

acrylamide is an accumlative neurotoxin and use a resipirator for TEMED

Pouring the gel

1.	Wash plates and spacers, rinse in distilled water and allow to dry.

2.	Degrease plates by wiping with ethanol, and when dry remove any dust with a fine brush.

3.	Put glass plates together with spacers about 2 mm in from edge.  Clip plates together.

4.	Prepare 25 ml 2% agar and melt.  Seal edges with agarose.

5.	Set up plates vertically.

6.	Make suffcient resolving gel mixture containing everything except the APS and TEMED, and de-gas for 10 min.

7.	Add APS and TEMED to degassed preparation, mix and dispense between plates. Avoid trapping air bubbles.

8.	Overlay with water saturated butanol, and allow to polymerise (10 min to 1 h)

9.	After polymerization of separation gel, pour away butanol and unpolymerized acrylamide, rinse with distilled water and allow to drain.  Use Whatman's No 1 paper to dry the top gel surface as much as possible.

10.	Make sufficient staking gel mixture (without APS and TEMED) and de-gas as before

11.	Add APS and TEMED, mix and dispense into avoiding air bubbles.

12.	Insert comb and allow to polymerize (about 1h).

Media, buffers and solutions	Form

resolving buffer
stacking buffer
acrylamide stock
ammonium persulphate, APS
gel mixes
TEMED


SDS-PAGE protein gels, preparing for electrophoresis
  SDS-PAGE gels

Related

Contributor:

Zappe, H

Reference:

-

Caution:

acrylamide is an accumlative neurotoxin and use a resipirator for TEMED

Preparing the gel for electrophoresis

1.	Remove clips from bottom-end of gel and CAREFULLY remove lower spacer.

2.	Remove comb CAREFULLY, wash wells and lower gel edge with distilled water.

3.	Place running buffer in lower buffer tank.

4.	Place gel into apparatus and secure with clips.

5.	Fill upper buffer tank to about 5mm above the gel plate.

6.	Remove bubbles trapped under gel, using siringe with curved needle.  Take care not to pry plates apart.

7.	Add samples with Hamilton syringe.Ensure EACH well is filled with the SAME volume.

8.	Plug leads into power pack

9.	Run the gel at 100 Volts to stack the proteins.  When the proteins enter the separating gel increase to 200 V (about 20 mA per gel).

Media, buffers and solutions	Form

tank buffer

sample buffer

acrylamide stock
gel mixes


SDS-PAGE protein gels, staining
  SDS-PAGE gels staining

Related

Contributor:

Zappe, H

Reference:

-

Caution:

acrylamide is an accumlative neurotoxin and use a resipirator for TEMED

Staining

????

Media, buffers and solutions	Form

stain


SDS-PAGE protein gels, drying
  SDS-PAGE gels drying

Related

Contributor:

Zappe, H

Reference:

-

Caution:

acrylamide is an accumlative neurotoxin and use a resipirator for TEMED

Gel drying

1.	Destained gels should be washed AT LEAST 4 times in 10% acetic acid to remove ethanol.

2.	Prevent gel cracking by soaking gel in 10% acetic acid containing 1% glycerol for 45 min prior to drying.

3.	Transfer gel on filter paper to dryer heating block, and remove any air bubbles trapped between the gel and the paper before drying is commenced.

4.	Check the red silicone sheet of the gel dryer for cracks before use.

5.	Cover gel with red silicone sheet and connect the vacuum pump.

6.	Turn on the heating element by turning on the timer.  Dry for about 60 min.

7.	Disconnect vacuum tube from dryer before opening red silicone sealing sheet.

Media, buffers and solutions	Form

destain


Recipe for resolving gel buffer
  Lower (resolving) gel buffer

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

RT

Lower (resolving) gel buffer

Component 	stock conc	final conc.	/ 200ml

Trizma base	1.5M	36.35g

SDS	0.4%(w/v)	0.8g

water	to 200ml

Adjust to pH 8.8 with concentrated HCl.


Recipe for stacking buffer
  Upper (stacking) gel buffer

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

RT

Upper (stacking) gel buffer

Component 	stock conc	final conc.	/ 200ml

Trizma base	0.5M	12.1g

SDS	0.4%(w/v)	0.8g

water	to 200ml

Adjust to pH 6.8 with concentrated HCl


Recipe for acrylamide stock solution
  Acrylamide-bis-acrylamide stock solution

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

in dark

Acrylamide-bis-acrylamide stock solution

Component 	stock conc	final conc.	/ 100ml

Acrylamide	29.2 g

bis-Acrylamide	0.8 g

water	to 100ml

Filter through Whatman's No1 paper and store in a dark bottle at 4 °C


Recipe for Ammonium persulphate
  Ammonium persulphate

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

Make fresh weekly

Ammonium persulphate (APS)

Component 	stock conc	final conc.	/ 0.5ml

APS	10%(w/v)	0.05g

water	0.5ml


  TEMED

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

4°C

Caution:

  use resipator

Used undiluted stock


Recipe for tank buffer (SDS-PAGE)
  Tank buffer

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

-

Electrophoresis (or tank) buffer  (10X)

Component 	stock conc	final conc.	/ 500ml

Trizma base	15.15 g

Glycine	72.05g

SDS	5.0g

water	to 100ml


Recipe for sample buffer, SDS-PAGE gels
  Sample buffer (SDS-PAGE)

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

-20°C

Sample treatment buffer

Component 	stock conc	final conc.	/ 100ml

Stacking gel buffer	29.2 g

SDS 	10%	4ml

Glycerol	2ml

2-mercaptoethanol	1ml

water	0.5ml


Recipe for acrylamide gel mixes
  Acrylamide gel mixes

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

-

Caution;

 acrylamide and TEMED are dangerous

Gel Mixtures

Component 	resolving gel	stacking gel	(7.5 %)	(12.5 %)

Resolving gel buffer	1.25 ml	1.25 ml	   --

Stacking gel buffer	    --	     --	1.25 ml

water	2.5 ml	1.65 ml	3.0 ml

Acrylamide Solution	1.25 ml	2.05 ml	750 μl

APS	15 μl	15 μl	15 μl

TEMED	10 μl	10 μl	10 μl

????????????????????


Recipe for SDS-PAGE gel stain
  SDS-PAGE gel stain

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

RT

SDS-PAGE gel stain

Component 	stock conc	final conc.	/ 500ml

Coomassie Brilliant Blue -R250	1.25 g

methanol	227 ml

water	227 ml

Glacial Acetic Acid	46 ml

Dissolve stain in methanol, add water and mix. Finally add acetic acid. Leave to stand overnight and filter.  The above volume of stain can be used for about 8 gels.


Recipe for SDS-PAGE gel destain
  SDS-PAGE gel destain

Contributor:

Zappe, H

Related:

SDS-PAGE gels

Storage:

RT

SDS-PAGE gel destain

Component 	stock conc	final conc.	/ 500ml

Ethanol	100%	20%

glacial acetic acid	10%

water	to 500ml

????????


Southern blots (Zappe), description
  Southern transfer of DNA (Zappe)

Related	Other Southerns

Description:

Southern transfer of DNA and blots (radioactive 32P).  Harold's method.

The protocol can be sub-divided into various stages.  USe the browse buttons to go through them, or click on the related topics button, or pick from below.

1.	Pretreatment

2.	Transfer

3.	Nick translation

4.	Spin column purification

5.	Wash/hybing

6.	Autorad


Southern blots (Zappe), pre-treatment
  Southern blots: Pre-treatment

Related

Contributor:

Zappe, H

Reference:

Smith and Summers, (1980),  Anal. Biochem. 109:123-129

Pre-transfer treatment

1.	Run Tris-acetate agarose gel of DNA samples over night to improve resolution.  Load 1 - 5 μg total digest of chromosomal DNA per lane for homologous hybridization to clone; 100 - 200 ng per lane for plasmid to plasmid hybridization depending on the number of bands.  Run λ DNA markers on one of the side lanes.

2.	Stain with EtBr and take a polaroid picture of the gel using type 665 Pos/Neg film: approx 80s exposure using the 254nm UV light source.  This gives a very good quality print and a negative for future printing but, more importantly, the irradiation of the DNA causes breaks which aids in the more efficient transfer of larger fragments.  Using a piece of paper alongside the gel, make a template of the markers.  The blot will be the same size as the gel, and the size template can be used as a reference when the hybridization is complete.  Alternatively, run DIG-labelled standards.

3.	Soak the gel twice for 15 min in 2-3 gel volumes of depurination solution (ST1) at room temperature with gentle shaking, to partially hydrolyse the DNA by acid depurination.  This causes double-stranded breaks in the DNA and aids transfer of large fragments (=20kb). Do not overdo!.  Rinse with dH2O.

4.	Soak the gel twice for 15 min in 2-3 gel volumes of denaturation solution (ST2) at room temperature with gentle shaking (to denature the DNA.).  Rinse with dH2O

5.	Soak the gel twice for 30 min in 2-3 gel volumes of neutralizing solution (ST3) at room temperature with gentle shaking (to neutralize).  Rinse with dH2O.

6.	Continue with either DNA transfer .

Media, buffers and solutions

depurinate solution (ST1)

denaturation solution (ST2)

neutralizing solution (ST3)


Southern blots (Zappe), mono-directional transfer
  Southern blots: Mono-directional transfer

Transfer        Southern

Contributor:

Zappe, H

Reference:

Smith and Summers, (1980),  Anal. Biochem. 109:123-129

Mono-directional transfer

1.	Place the gel on a piece of glass on a flat surface and dab dry with paper towel.

2.	Cut off all unnecessary peices of gel and cut one corner to serve as a reference.  Then cut a piece of Hybond-N+ to fit (handle only with a piece of protective paper on each side or use tweezers - never touch it!) and float it onto the ST3 solution wash to wet, then allow to soak for about 2 min.

3.	Layer the membrane onto the gel being sure to eliminate all air bubbles.

4.	Wet 3 pieces of Whatmans No 1 filter paper in the same solution and layer them on top of the Hybond-N+.

5.	Place about 30 - 50 mm of dry paper towels on top, followed by another piece of glass and about a 2 kg weight to compress the sandwich.

6.	Allow to transfer for 60 - 90 min.

7.	Peel everything off, and remove the Hybond-N+ filter for alkali fixation

8.	Place the membrane on 3 layers of absorbent filter paper (Whatmans 3MM) soaked in 0.4 M NaOH.  The manufacturer suggests a fixing time of 2 - 60 min, but recommends 20 min.

9.	Rinse the membrane briefly with 5X SSC with gentle shaking  for 1 min.  Seal the membrane in a plastic bag and continue with hybridisation or store at 4 °C.

Media, buffers and solutions

SSC


Southern blots (Zappe), bi-directional transfer
  Southern blots: Bi-directional transfer

Related        Southern (Zappe)

Contributor:

Zappe, H

Reference:

Smith and Summers, (1980),  Anal. Biochem. 109:123-129

Bi-directional transfer.

1.	Wet both Hybond-N+ membranes and filter paper in ST3.

2.	Place 3 pieces of Whatmane No.1 filter paper on a glass plate, then a piece of Hybond-N+, followed by the gel, more Hybond-N+ and filter paper as for mono- transfer.

3.	Lift this sandwich from the glass plate and place on 30mm of paper towel. Place a further stack of paper towel on top, followed by a glass plate and the weight.

4.	Allow to transfer for 60 - 90 min., and then continue with alkali fixing with both membranes.

Media, buffers and solutions

ST3 neutralizing solution


Southern blots (Zappe), nick translation
  Southern blots: Nick translation

Related        Southern (Zappe)

Contributor:

Zappe, H

Reference:

Rigby et al., 1977. J. Mol. Biol. 113:237.

Caution:

 Full radiation safety protocols

Nick-translation

1.	The nick-translation Amersham kit uses a 100 μl reaction and 10 μl of 32P label which is wasteful and unnecessary!  The reaction can be scaled to half or to 1/4 of these volumes and still produce an excess of labelled probe.

2.	Thaw the kit buffers and hold on ice.  Always keep the enzyme solution on ice.

3.	Combine the following in an eppendorf in order:

5 μl DNA  (0.3-1 μg)

5 μl kit solution 1

2.5 μl kit solution 2

10 μl water

2.5 μl 32P dN(A or C)TP

4.	Incubate for 2 h at 15 °C.

5.	One can do TCA precipitable (Sambrook et al., Appendix E, page 18) counts on the reaction to obtain accurate % incorporation (eg. for a new kit), but normally this is not necessary.

6.	Make up the volume of the nick-translation to 100 μl by adding the appropriate amount of STE

7.	Remove 1 μl to a scintillation vial (eject tip into the vial) for a total count control.

8.	Pass the rest of the sample through a spin coloumn, and again remove 1 μl to determine the incorporated counts.  Calculate the total counts recovered and compare with the control for % incorporation.  Should be 50 % or more. (incorporation can vary depending on labelled nucleotide used vs GC content of the DNA).  This method is not as good as TCA's but is quick and good enough.  As some of the DNA remains on the coloumn after 1 pass the % calculated is usually an underestimate.

Media, buffers and solutions

STE


Southern blots (Zappe), spin column purification
  Southern blots: Spin coloumn purification

Southerns	DNA purification

Contributor:

Zappe, H

Reference:

Sambrook et al. Appendix E, p 37.

Spin coloumn purification

(removal of un-incorporated dNTP's from the probe solution)

1.	The method used is exactly that described in Sambrook et al. Appendix E, p 37.

2.	Use a 15 ml Falcon tube to hold the syringe.

3.	Make the plug from siliconized sterile glass wool and use the plunger to position it at the bottom.

4.	Do all spins at 55 % (about 1200 RPM) on the swing-out rotor for 4 min.

5.	After spinning the probe through, one can wash the coloumn with 100 μl STE and recover a bit more probe, but keep this separate from the rest (the nucleotides only start coming out on the third or fourth wash).

Media, buffers and solutions


Southern blots (Zappe), hybridization and washing steps
  Southern blots: Hybridization/washing (Zappe)

Related

Contributor:

Zappe, H

Reference:

Both Sambrook et al. and the book "Nucleic acid Hybridzation.  A Practical Approach" (edited by Hames and Higgins) discuss all aspects of hybridization - good reading!!.

Hybridization of probes to DNA bound to Hybond-N+/ Nitrocellulose filters.

1.	The method is essentially the same as Sambrook et al. but with a few speeded up steps.  The steps are described in Sambrook et al. page 9.47 - 9.54.

2.	Make up the Prehybridization/Hybridization solution:

3.	Float the filter on 6 X SSC to wet, then soak for 2 min.

4.	Place the filter in a plastic bag, add 75 μl/cm2 prewarmed hybridization fluid, remove all air, seal the bag and prehybridize for 1 to 3 h, with gentle shaking at the hybridization temperature. (Calculate this temp. using the formula on pg 9.50 point 11A).  Remove as much air from the bag as possible.

Denaturing the probe

1.	Make a solution of probe and TE to a final vol. of 100 μl, seal the Eppendorf with tape and boil for 5 min then cool immediately on ice.

2.	The alkali denaturing method of Meinkoth and Wahl (1984) (Anal. Biochem. 138:267) can also be used.

5.	Cut off a corner of the bag, add the denatured probe, reseal, and hybridize with shaking at the same temp. for 16 h (O/N).

6.	Add about 1 x 107 CPM for the probe.

Washing the filter after hybridization.

The stringency of the washing steps determines the sensitivity of the method.  If the probe is not degenerate, then high stringency washes are used.

1.	Wash twice at R/T with 2 X SSC, 0.5% SDS to rinse off unbound probe.

2.	Wash for 15 min at 40 °C in 0.1 X SSC, 0.1% SDS.  Check the counts on the filter: Areas where no counts are expected should be clean, otherwise wash again.

3.	Increase the temperature for further washes if the backgroung is a problem.

Media, buffers and solutions

prehyb solution

SSC

SDS


Southern blots (Zappe), autoradiography
  Southern blots: autoradiography

Related

Contributor:

Zappe, H

Reference:

Smith and Summers, (1980),  Anal. Biochem. 109:123-129

Setting up the autorad.

1.	After the last wash seal the filter in a plastic bag (do not let it dry!) free of air bubbles, place a film on each side in a cassette and store O/N at -70 °C.  Develop one film the next day: if underexposed, leave the second one another 24 h.

2.	If the background is too high, remove the filter from the bag and wash again, increasing the stringency.

3.	The filter can be used with a another probe if the first probe is stripped off by washing the filter for about 10 min in water at > 80 °C.


Recipe for Southern transfer solution 1
  Depurination solution (ST1)

Contributor:

Babb, B

Related:

Southern transfer DNA

Storage:

RT

Depurination solution (ST1)

Component 	stock conc	final conc.	/ 200ml

HCl	conc.	0.25M	??

water	to 200ml


Recipe for Southern transfer solution 2
  Denaturnation solution (ST2)

Contributor:

Babb, B

Related:

Southern transfer DNA

Storage:

RT

Denaturation solution (ST2)

Component 	stock conc	final conc.	/ 200ml

NaOH	10M	0.5M

NaCl	5m	1.5M

water	to 200ml


Recipe for Southern transfer solution 3
  Neutralizing solution (ST3)

Contributor:

Babb, B

Related:

Southern transfer DNA

Storage:

RT

Neutralizing solution (ST3)

Component 	stock conc	final conc.	/ 200ml

Ammonium acetate	1M

NaOH	0.02M

water	to 200ml


Recipe for STE
  STE

Contributor:

Related:

-

Storage:

RT

STE

Component 	stock conc	final conc.	/ 200ml

Tris buffer, pH 8.0	1.0M	10mM	2ml

EDTA, pH 8.0	0.5M	1mM	0.4ml

NaCl	5M	100mM	4ml

water	to 200ml


Recipe for hybridization solution (Zappe)
  Pre-hyb/hyb solution

Contributor:

Zappe, H

Related:

Southern transfer DNA

Storage:

RT

Component 	stock conc	final conc.	/ 200ml

9 ml 20 X SSC

0.6 ml 25% SDS

1.2 ml BLOTTO ( Sambrook et al., page B15)

0.3 ml 10 mg/ml boiled Salmon sperm DNA

18.9 ml water

water	to 200ml


Protein, Western, description
  Western blots

Westerns

Description:

Western blotting from SDS-PAGE gels onto nitrocellulose membranes is generally done using the method of Towbin et al. (1979).

Zappe uses the Boehringer Mannheim detection system.  See here


Proteins, western blots (Zappe), protocol outline
  Western blots (Zappe)

Related

Contributor:

Zappe, H

Reference:

Towbin et al. (1979)., Boehringer Mannheim protocols

1.	Set up the transfer apparatus as described in the manufacturers instructions.

2.	Electroblot onto a nitrocellulose membrane in blot buffer at 100 V (constant voltage) for 2 h using a Hoefer Transphor (model TE 42) (Hoefer Scientific Instruments, San Francisco).

3.	The method of detection used is the BM DIG system, a protocol of which is attached.

Media, buffers and solutions

Blocking buffer stock solution

Blocking buffer

Blot buffer


Recipe for blocking buffer stock solution
  Blocking buffer stock solution

Contributor:

Zappe, H

Related:

Western blots (Zappe)

Storage:

RT

Blocking buffer stock solution

Component 	stock conc	final conc.	/ 1000ml

Tris-Cl  (pH 7.4)	1.21 g

NaCl	9 g

Distilled water	 to 1000 ml


Recipe for blocking buffer
  Blocking buffer

Contributor:

Zappe, H

Related:

Western blots (Zappe)

Storage:

RT

Blocking buffer

Component 	stock conc	final conc.	/ 1000ml

Blocking buffer stock solution	500 ml

Fat-free milk powder	10 g

Tween-20	0.25 ml

Sodium azide	0.1 g

Distilled water	 to 1000 ml


Recipe for blot buffer
  Blot buffer

Contributor:

Zappe, H

Related:

Western blots (Zappe)

Storage:

RT

Blot buffer

Component 	stock conc	final conc.	/ 5000ml

Tris base	25 mM	15 g

Glycine	0.192 M	72 g

Methanol	20%, v/v	1000 ml

Distilled water	to 5000 ml


REcipe for BSA solution
  Bovine Serum Albumin (1%)

Contributor:

Coyne, V

Related:

-

Storage

-20°C

Bovine Serum Albumin (1%)

Component 	stock conc	final conc.	/ 10ml

BSA (Pentax Fraction V)	1%	100mg

water	to 10ml

Filter sterilise, aliquot into sterile eppendorfs and store at -20°C.


Recipe for CTAB/NaCl solution
  CTAB/NaCl solution

Contributor:

Coyne, V

Related:

DNA extraction

Storage

CTAB (hexadecyltrimethyl ammonium bromide)/ NaCl solution

Component 	stock conc	final conc.	/ 100ml

NaCl	4.1g

water	80ml

add slowly, while heating (up to 65°C) and stirring:

CTAB 	10g

water	to 100ml


Recipe for Tris-Base (1M)
  Trisbase (1 M)

Contributor:

Coyne, V

Related:

-

Storage

Trisbase (1 M)

Component 	stock conc	final conc.	/ 100ml

Tris	1M	12.1g

water	to 100ml


Recipe for E. coli minimal medium
  E. coli minimal medium

Contributor:

Coyne, V

Related:

-

Storage

E. coli minimal medium

Component 	stock conc	final conc.	/ 100ml

distilled water	60 ml

agar	1.5 g

5 X salts	20 ml

distilled water	20 ml

glucose	20%	1 ml

MgSO4 	20%	0.1 ml

vitamin B1	0.5%	0.1 ml

amino acids 	* 	0.1 ml

* Amino acids:	20 mg/ml for Lamino acids and 40 mg/ml for DLamino acids.

The agar and salt solutions are autoclaved separately, cooled to 50°C,  combined and only then the rest of the constituents added.


Recipe for 5 x salts (minimal medium)
  5 X Salts stock

Contributor:

Coyne, V

Related:

E. coli minimal medium

Storage

do not autoclave, store over chloroform at 4°C

5 X Salts stock

Component 	stock conc	final conc.	/ 1000ml

KH2PO4	52.5 g

K2HPO4	22.5 g

ammonium sulphate, (NH4)2SO4	5.0 g

Sodium citrate	2.5 g

water	to 1000ml


Large scale preparation of bacterial genomic DNA
  Genomic DNA prep

Related        Description

Contributor:

Coyne, V

Reference:

adapted from Current Protocols, unit 2.4

Large Scale Preparation of Bacterial Genomic DNA

 1.	Grow 100 ml culture of bacterial strain to saturation.

 2.	Spin at 6K for 10 min and discard the supernatant.

 3.	Resuspend the pellet in 9.5 ml TE buffer, 0.5 ml of 10% SDS, and 50 ul of 20 mg/ml proteinase K.  Mix and incubate 1 h at 37°C.

 4.	Add 1.8 ml of 5 M NaCl. Mix thoroughly.

This step is very important since CTAB-nucleic acid precipitate will form if the salt concentration drops below 0.5 M at room temperature.  The aim here is to remove cell wall debris, denatured protein, and polysaccharides complexed to CTAB, while retaining the nucleic acids in solution.

 5.	Add 1.5 ml CTAB/NaCl solution and mix.  Incubate 20 min at 65°C.

 6.	Extract thoroughly with an equal volume of chloroform/isoamyl alcohol.  Spin 10 min at 7 K at room temperature, to separate the phase.

This extraction removes CTAB-protein/polysaccharide complexes. A white interface should be visible after centrifugation.

 7.	Transfer aqueous supernatant to a fresh tube.  Repeat step 6 if necessary.

 8.	Transfer aqueous phase to a fresh tube.  Precipitate DNA with 0.6 volume of isopropanol (there is no need to add salt since the NaCl concentration is already high), and mix until a stringy white DNA pellet precipitates out of the solution and condenses into a tight mass.  Transfer the precipitate to 1 ml of 70% EtOH in a fresh tube by hooking it on the end of a Pasteur pipette that has been bent and sealed in a Bunsen flame.  Spin the pellet 5 min at 9 K.  Remove the supernatant and dry under vacuum (do not overdry as DNA willl take longer to resuspend).

 9.	Resuspend the DNA pellet in 4 ml of TE buffer.  This may take several hours to overnight at 4°C.

10.	Measure DNA concentration on a spectrophotometer.

Critical parameters:

The most critical parameter is the salt concentration of the solution containing the lysed bacteria prior to adding CTAB.  If the NaCl concentration is <0.5 M, then the nucleic acid may also precipitate; indeed, CTAB is frequently used for just this purpose. It is also important to maintain all solutions above 15°C, as CTAB will precipitate below this temperature.

Media, buffers and solutions

TE buffer

SDS (10%)

proteinase K, 20mg/ml, (stored at 20°C);

NaCl;

CTAB/NaCl solution;

24:1 chloroform/isoamyl alcohol;

isopropanol

ethanol 70%


Electroelution of DNA from gels
  Electroelution from agarose gels

Related methods        Description

Contributor:

Coyne, V

Reference:

modified from Selden and Chory 1987. Current Protocols in Molecular Biology, unit 2.6.1

Used for isolating DNA restriction fragments from agarose gels.  The technique is particularly appropriate for purifying larger amounts (>500 ng) of fragments.

 1.	Digest sufficient DNA with the appropriate enzymes.

The relative abundance of the DNA fragment to be isolated in the sample to be electrophoresed, and the use to which this fragment will be put, are the important factors in determining the initial amount of DNA.  Try to aim for ~1 ug of DNA in the target band (sufficient for cloning experiments):

ie.	if the target band is 2.8 kb and the DNA sample is 10.3 kb (target = 27% of sample), then the amount of DNA which must be digested will be 3.7 μg (10.3/2.8)

 2.	Load sample onto the appropriate agarose gel and electrophorese.

Use a wide slot (4-5 cm) on a large gel when large amounts of DNA are to be separated or when the DNA fragment is difficult to resolve from the other fragments, while a minigel is best when a small amount of a well-separated fragment is to be isolated.

 3.	After electrophoresis stain the gel with ethidium bromide and photograph.

 4.	Carefully cut out the target band using a scalpel.

Try to minimise the time of exposure to UV light - especially if the DNA is to be used in cloning experiments.  Use long wavelength UV light to minimise DNA damage.

 5.	Rinse dialysis tubing (Spectra/por dialysis membrane tubing #4) with TAE buffer for 5 min.  Tie off/clip one end of the tubing and slide the gel slice into the tube.  Fill the tube with TAE buffer (~500 ul) until it is almost completely expanded and then knot/clip the other end.

 6.	Place the sealed bag in a gel box and fill the box with 1xTAE until the bag is just covered by the solution.  Make sure that the bag is parallel to the electrodes, and that the gel slice is against the side of the tubing closest to the negative electrode.

 7.	Electroelute at a constant voltage of ~2 V/cm between the 2 electrodes.

For a 500 - 2 000 bp target fragment - electroelute for 2 hr.  For a 2 - 4 kb fragment - electroelute at 1 V/cm overnight.  Never allow the running buffer to heat up as this will reduce yield significantly.

 8.	After electroelution is complete, reverse polarity of the electrodes and turn on voltage at 100 V for 30 sec.  This will free any DNA that has stuck to the dialysis tubing.

 9.	Carefully open one end of the tubing and collect the TAE buffer with a pasteur pipette and place in a microfuge tube.

The gel slice may be restained with ethidium bromide to ensure that all the DNA has been eluted.  If DNA still remains in the gel, go back to step 5 and repeat the elution.

10.	The DNA can now be concentrated by ethanol precipitation and resuspended in the appropriate volume of TE buffer.

Media, buffers and solutions


Geneclean of DNA ragments from agarose gels
  Geneclean of DNA from gels

Related methods        Description

Contributor:

Coyne, V; Zappe, H

Reference:

-

1.	Cut out band in as small a volume as possible.

i)	If using LMP, melt at 70°C, then determine volume.

ii)	If using ordinary agarose, spin down and determine volume by comparison with water in a similar tube, or weigh the gel slice and accept that 0.1 μg - 100 μl.

2.	Add 2.5 volumes of saturated SodiumIodide (NaI) solution.  Mix and hold at 50°C for 1 min, mix again, and leave at 50°C for 5 min.  Then check gel dissolved  if not incubate further.

3.	Add glassmilk:  5 μl if [DNA] < 5μg ; 2 μl for each additonal μg above 5 μg.

4.	Mix well and leave on ice for 5 min.

5.	Spin for 5 seconds, discard supernatant.

6.	Wash pellet 3 times with 500 μl ice cold NEWbuffer (10  50 vol).  Do this by resuspending the pellet each time with a Gilson pipette.

7.	Spin as before and remove NEWbuffer.

8.	Finally spin to collect last drops at the bottom, and remove.

9.	i.)	Elute DNA from glassmilk by dissolving pellet in 1  2 vol TE (1  2 vol of the glassmilk added i.e. 5  10 μl).

ii.)	Hold at 50°C for 2  3 min, then spin 5 sec and carefully recover TE containing DNA.

10.	Repeat elution and pool with first recovery (recover DNA in about 20 μl).

Media, buffers and solutions

NEW buffer

TE buffer

glassmilk


  Geneclean of DNA from reactions

Related methods        Description       DNA reactions

Contributor:

Coyne, V; Zappe, H

Reference:

-

1.	Add 2.5 vol NaI stock and mix.

2.	Add glassmilk (5 μl for 5 μg DNA + 2 μl/μg above 5 μg) and leave 5 min on ice.

3.	Spin 5 sec and discard supernatant.

4.	Wash 3 times with ice cold NEWbuffer.

5.	Elute DNA as above to TE or water.

Media, buffers and solutions

NEW-buffer

Sodium iodide (NaI)

TE buffer

glassmilk


DNA precipitation by isopropanol/sodium perchlorate, protocol
  Isopropanol Precipitation

Related methods        DNA reactions

Contributor:

Zappe, H

Reference:

-

1.	Add 1/10 volume of 5 M NaClO4 and an equal volume of isopropanol.  Mix, leave on ice for 5 min and then spin for 10 min.

2.	Wash the pellet with 70 % EtOH and spin 2 min with tube re-orientation.  Pour off EtOH, spin to pellet the last drops and draw off with a P200 pipette.

3.	Resuspend in water or TE depending on the following reaction.

Media, buffers and solutions

NaClO4

Isopropanol

TE buffer

ethanol 70%


DNA precipitation by ethanol/ammonium acetate, protocol
  Ammonium acetate precipitation of DNA

Related methods        DNA reactions

Contributor:

Coyne, V

Reference:

-

1.	Heat inactivate the enzyme to stop the reaction reaction by incubation at 65°C for 10 min.

Not all restriction enzymes can be denatured by heat; consult supplier's manual.  Often enzymes that are partially, or completely, resistant to inactivation at 65°C can be denatured at 75°C for 15 min.  Alternatively, the enzyme is denatured using phenol - chloroform prior to precipitation with ammonium acetate (sectn. III., steps 1-5).

2.	Add 0.5x volume 7.5 M NH4 acetate (pH 7.5) to the DNA suspension.

3.	Incubate at room temperature for 30 minutes.

4.	Microfuge at room temperature for 15 minutes.

5.	Transfer the supernatant to a new eppendorf tube.

6.	Add 2.5x volumes 100% ethanol and incubate at room temperature for 0 min to overnight (depending on concentration of DNA; see Table below).

7.	Microfuge for 30 min at room temperature.

8.	Wash pellet with 70% ethanol (take care not to throw out the pellet with the ethanol; pour out gently and aspirate the remaining ethanol).

9.	Dry pellet and resuspend in the required volume of TE buffer.

NOTE:	2 - 3 cycles of the above protocol will yield "squeaky" clean DNA.  Thus, this method is recommended for cleaning up DNA preps which won't cut to completion etc.  The first step removes protein contaminants, while the second step concentrates the DNA.

Effect of time on ethanol precipitation with ammonium acetate at 22°C.

Percent DNA Recovered

DNA conc.	0 min	10 min	30 min	overnight

5 μg/ml	88	97	93	100

0.5 μg/ml	62	64	65	92

0.05 μg/ml	47	40	36	87

0.005 μg/ml	40	35	38	85

Media, buffers and solutions

ammonium acetate

ethanol


Dephosphorylation of DNA, description
  Dephosphorylation of DNA

Related methods        DNA reactions

Contributor:

Coyne, V

Reference:

-

Bacterial alkaline phosphatase (BAP) and calf intestine phospahatase (CIP) are routinely used to remove the 5' PO4 groups from linearised DNA.  This treatment prevents linearised vector DNA from recircularising when carrying out intermolecular ligation reactions with foreign DNA.  This is important, as vector religation results in large numbers of transformants which merely contain parental plasmid DNA instead of the required recombinant molecules; thus necessitating laborious screening of transformants for the desired clone.

1.	Typical CIPping reaction (Coyne)


Cipping of vector DNA, protocol
  Cipping of Vector DNA

Related methods        DNA reactions

Contributor:

Coyne, V

Reference:

-

For a typical CIP reaction:

vector DNA 1 to 20 pmol ends	x ul

1 M Tris, pH 8.0	1 ul

50 mM MgCl2	1 ul

50 mM ZnCl2	1 ul

0.1 U CIP	y ul

dH2O	z ul

Total treaction volume	50 ul

Incubate at 37°C for 30 min.  Stop the reaction by heating at 75°C for 10 min, ethanol precipitate DNA to change to ligation buffer.

The phosphatase treatment can be done directly following restriction enzyme cleavage by merely adding the Mg2Cl, ZnCl2, and Tris, and making the volume up to 50 ul.  This will minimise the number of ethanol precipitations which can lead to loss of DNA.


DNA ligation, hints for troubleshooting (Coyne)
  Ligation hints/ troubleshooting

Related

Contributor:

Coyne, V

Reference:

-

Hints for reducing transformation background:

1.	Wherever possible use 2 different enzymes to linearize the vector and insert DNA to prevent vector religation.

2.	If this is unavoidable, use dephosphorylated vector at a ratio of vector:insert of 4:1.

3.	Use biochemical selection to prevent cloning of unwanted fragments; ie. insertional inactivation of a lethal gene carried on the vector (eg. pEcoR251).

Troubleshooting suggestions (controls):

1.	Try a series of simple control ligation experiments where you linearize the vector with the restriction enzyme for various times at various enzyme/vector ratios.  Then just do a transformation without the ligation step.  This will help you determine the optimum digestion conditions to minimize any uncut vector.

2.	Once you've found digestion conditions where there is minimum uncut vector, do a time study for BAP or CIP dephosphorylation (usually need 30min, 1hr, 2 hr).  Do a ligation/transformation for each time point without added insert to determine the efficiency of the phosphatase treatment.

3.	Repeat the ligation/transformation using the dephosphorylated vector with minimal background produced in step #2 and this time use varying amounts of insert for the ligation, holding the vector amount constant.

Troubleshooting (reactions in gels)

1.	The larger bands in any DNA gel lane always contain a small percentage of the fragments migrating in front (faster) than them.  This is because the faster bands actually tail throughout the lane.  Thus, if the smaller bands have the same cohesive ends, expect to find some clones containing these fragments, or use biochemical selection.

2.	There is no difference whether one treats with CIP or BAP before or after gel purification.

3.	The only enzyme that does not work in gel containing solutions is T4 polynucleotide kinase.


Recipe for SSC 20x
  SSC 20x

Contributor:

Coyne, V

Related:

-

Storage

Salt sodium citrate, SSC 20x

Component 	stock conc	final conc.	/ 100ml

NaCl	17.5 g

tri-sodium Citrate	8.82 g

water	80 ml

Adjust pH with 10 N NaOH to 7.4

water	to 100 ml

Autoclave

For 2x SSC:
dilute 20ml of 20x in 180ml water

For 0.1 x SSC:
dilute 1ml of 20x in 199ml water


Recipe for denaturation solution (0.4M NaOH, 1M NaCl)
  Denaturation solution (Coyne)

Contributor:

Coyne, V

Related:

Southern blots

Storage

0.4 M NaOH/ 1 M NaCl

Component 	stock conc	final conc.	/ 1000ml

NaOH	0.4M	16 g

NaCl	1M	58.44 g

water	to 1 l


Transfer of DNA to membrane, protocol (Coyne)
  Southern Transfer of DNA

Related

Contributor:

Coyne, V

Reference:

(modified from Reed & Mann 1985. NAR 13: 7207-7221)

1.	Soak gel in 2 X vol. 0.25 M HCl with agitation for 5 min @ room temp. (or until stop buffer turns yellow).

2.	Rinse in dH2O

3.	Saturate 10 sheets (25 x 20 cm) Whatman 3MM paper with 0.4 M NaOH.  Place on inverted gel casting tray which has been placed in a tray covered with Saran wrap.  Add 0.4 M NaOH/1 M NaCl to tray so that the ends of the wicks (Whatman paper) are submerged.

4.	Invert gel and place on top of saturated Whatman paper. Make sure that no air bubbles remain trapped.

5.	For gel = 14.5 cm x 19.5 cm, cut nylon membrane (Hybond N+) 15 cm x 20 cm.  Wet membrane in dH2O, soak in 0.4 M NaOH/1 M NaCl for 5 min & place on gel.  Check that no air bubbles remain trapped.  Cover edges with Saran wrap on tray.

Do not handle membrane without gloves.

7.	Cover the membrane with 3 X sheets (11.2 cm x 14.9 cm) of Whatmans 3MM paper (wet 1st sheet with dH2O and blot off excess fluid, place on gel - ensure that no air bubbles remain trapped, place remaining 2 sheets of dry filter paper on top of wet sheet), followed by a 3 inch stack of dry absorbent paper towel.  Place a glass plate on top of the towels, followed by a 0.2 - 0.4 kg weight.

8.	Blot for 1 hr. - O/N.  Usually 2 - 4 hr sufficient.

9.	Mark wells on membrane with pencil & rinse in 2x SSC for 2 - 5 min.

10.	Air dry membrane on dry Whatman paper for 30 min.  Store between 2 X sheets dry Whatman paper.

Media, Buffers, solutions

0.25M HCl

0.4M NaOH/1M NaCl

SSC (2x)


Recipe for Sephadex G-50
  Sephadex G-50

Contributor:

Coyne, V

Related:

-

Storage

4 °C

Sephadex G-50

Component 	stock conc	final conc.	/ 100ml

add slowly:

Sephadex G-50	30g

to

STE	250ml

Make sure the powder is well dispersed when adding the Sephadex G-50 to the STE

Autoclave - swells the beads.


Recipe for tracking dye (Coyne)
  Tracking dye (Coyne)

Contributor:

Coyne, V

Related:

-

Storage

4 °C

Tracking dye

Component 	stock conc	final conc.	/ 10ml

NaCl	5M	50mM	100 μl

water	8ml

Blue dextran	3%	0.3g

Orange G	1%	0.1g

water	to 10ml


DNA purification by spin column (Coyne), protocol
  Spin column

Related       Southerns

Contributor:

Coyne, V

Reference:

Modified from Cold Spring Harbour Manual

This protocol is used to separate radioisotope-labelled DNA from unincorporated nucleotides.

1.	Plug the bottom of a 1 ml disposable syringe with a small amount of sterile glass wool.

2.	In the syringe, prepare a column (0.9 ml bed volume) of Sephadex G-50 equilibrated in STE buffer by adding Sephadex to the top of the syringe and then placing the syringe in a disposable bench top centrifuge tube in a benchtop centrifuge.

3.	Centrifuge the tube for 1 min. at 1600g (setting 40% on orange benchtop centrifuge = 4 000 U/min).

4.	Repeat steps 2 & 3 until a column bed volume of 0.9 ml is obtained.

5.	Add 0.1 ml of STE to the column and recentrifuge at the same speed as in #3.

6.	Prepare labelled DNA sample by adding 10 ul tracking dye and 40 ul STE buffer (total volume of 100 ul).

7.	Place an eppendorf tube (with cap removed) at bottom of benchtop centrifuge tube so that the syringe will empty into the eppendorf tube.

8.	Load the DNA to the column and centrifuge for 4 min. The tracking dye will move with the sample as follows: the Blue Dextran will move with the DNA probe, while the Orange G will migrate with the unincorporated nucleotides on the column.  Thus, it is possible to visually monitor the elution of the probe DNA; ie. additional centrifugation for periods of 1 min. may be carried out if necessary.

9.	Determine the specific activity of the labelled DNA by counting 1 ul of probe in 2 ml of scintillation fluid.

Specific activity = counts per minute (cpm)/ug DNA

Media, Buffers, solutions

STE

Sephadex G50

scintillation fluid


Recipe for phosphate buffer (PB)
  Phosphate buffer stock

Contributor:

Coyne, V

Related:

-

Storage

Phosphate buffer (PB), 1M, pH 7.2

Component 	stock conc	final conc.	/ 1l

Na2HPO4.7H2O	0.5 M	134 g

phosphoric acid,H3PO4 	85% 	4 ml

water	to 1l

Autoclave


Recipe for Church pre-hyb buffer, CHB
  Church Pre-Hybridization Buffer (CHB)

Contributor:

Coyne, V

Related:

Southern hybridization

Storage

Church Pre-Hybridization Buffer (CHB)

Component 	stock conc	final conc.	/ 100ml

Carnation Non-fat Dry Milk	0.5% 	0.5 g

dH2O	20.5 ml

PB 	1M	0.5 M 	50.0 ml

EDTA 	0.1M	1 mM 	 1.0 ml

SDS 	25%	7% 	28.0 ml

Church Pre-Hybridization Buffer (CHB)  = 1.2 M Na+


Recipe for Church hyb buffer
  Church Hybridization buffer

Contributor:

Coyne, V

Related:

Southern hybridization

Storage

Church Hybridization buffer

Component 	stock conc	final conc.	/ 100ml

PB 	1M	0.5 M 	50.0 ml

EDTA	0.1M	1mM	1.0 ml

SDS 	25%	7%	28.0 ml

water	20.5 ml


Recipe for wash buffer A WBA
  Wash Buffer A (WBA)

Contributor:

Coyne, V

Related:

Southern hybridization

Storage

Wash Buffer A (WBA)

Component 	stock conc	final conc.	/ 500ml

SDS	25%	5%	100ml

PB	1M	40mM	5ml

EDTA	0.1M	1mM	5ml

water	375ml

WBA = 0.25 M Na+


Recipe for wash buffer B WBB
  Wash Buffer B (WBB)

Contributor:

Coyne, V

Related:

Southern hybridization

Storage

Wash Buffer B (WBB)

Component 	stock conc	final conc.	/ 1l

SDS	25%	1%	40ml

PB	1M	40mM	40ml

EDTA	0.1M	1mM	10ml

water	to 1l

WBB = 0.11 M Na+    (~ 0.2X SSC)


Recipe for wash buffer C WBC
  Wash Buffer C (WBC)

Contributor:

Coyne, V

Related:

Southern hybridization

Storage

Wash Buffer C (WBC)

Component 	stock conc	final conc.	/ 200ml

SDS	25%	0.1%	0.8ml

PB	1M	20mM	4ml

EDTA	0.1M	1mM	2.0ml

water	to 200ml

Optional: very stringent conditions

WBC = 0.04 M Na+     (~ 0.1X SSC)


DNA-DNA hybridization by modified Church protocol (Coyne)
  DNA-DNA hybridization

Related

Contributor:

Coyne, V

Reference:

(Modified from Church & Gilbert 1984. PNAS 81: 1991-1995)

1.	Wet nylon membrane with CHB & place in plastic bag.

2.	Add 0.2 ml CHB per cm2 membrane.  Seal bag & incubate at 65°C for 10 min with agitation.

3.	Denature probe DNA by placing in a boiling water bath for 10 min and then cooling rapidly by placing directly on ice.

4.	Remove CHB.  Add fresh CHB to the bag (50 ul/cm2 membrane).  Add denatured probe DNA to the bag (use 1 -15 ng probe DNA per ml of hybridization buffer where the specific activity of the probe is from 5 x 107 cpm/ug to > 108 cpm/ug.  Aim for 5 x 105 - 1 x 106 cpm per ml of hybridization buffer).  Remove bubbles and seal bag. Incubate O/N at 65°C with agitation.

5.	Remove membrane.  DO NOT allow to dry!

6.*	Wash 2 X in WBA (prewarmed) at 65°C with agitation (10 min per cycle).

7.*	Wash 4 X in WBB (prewarmed) at 65°C with agitation (10 min per cycle).

*	Monitor counts between washes with Geiger counter.  Stop washing if most of membrane begins to count around background (between 200 - 500 cpm).

8.	Rinse briefly in dH2O to remove SDS.

9.	Wrap membrane with Saran wrap or seal in new plastic bag (do not allow to dry).

10.	Place sealed membrane in an autoradiograph cassette containing enhancer screens.  Place a sheet of X-ray film (either Kodak X-OMAT or XAR - use former as < expensive) over the bag containing the membrane - DO THIS IN THE DARKROOM WITH THE X-RAY SAFETY LIGHT ON. Close the cassette and place at -70°C for 60 min - to overnight (depends on amount of radioactivity bound to filter - homologous probes will be very "hot" and thus only require short exposure times).

11.	Thaw cassette for 30 min prior to developing film.  Open cassette in darkroom with X-ray safety light on and feed film into the automatic X-ray developer.  To develop manually, place film in Kodak X-ray developer for 5 min, in X-ray stop solution for 1 min, and in X-ray fixer for 5 min.  Wash for 10 min in water.  Air dry.

NOTE:

1.	Hybridization is performed under non-stringent conditions, while the post-hybridization washes are carried out at Tm -5°C (homologous) or Tm -15°C (heterologous).

2.	Tm = 81 + 16.6(log Na+) + 0.4(%G+C) -

where:

Na+ = molar concentration of sodium ions

n = length of the probe (insignificant for probes  < 600 bp

Media, Buffers, solutions

CHB

WBA

WBB

WBC


   PCR

A.	Applications

B.	Dealing with products

C.	Miscellaneous

D.	Primer design

E.	Reaction conditions

F.	References

G.	Sample reaction

H.	Sequencing PCR Products


   PCR

Applications

1.	Asymmetric PCR for ssDNA, description (Rybicki)

2.	cDNA PCR, description (Rybicki)


   PCR

Reaction conditions

1.	Annealing temperature and time, description (Rybicki)

2.	Cycle number, description (Rybicki)

3.	Denaturing temperature and time, description (Rybicki)

4.	Elongation temperature and time, description (Rybicki)

5.	Helix Destabilisers / Additives, description (Rybicki)

6.	"Hot Start" PCR:, description (Rybicki)

7.	Initial Conditions, description (Rybicki)

8.	Reaction buffer, description (Rybicki)

9.	Recommended Reagents, description (Rybicki)

10.	Temperature cycling, description (Rybicki)


   PCR

Primer design

1.	simple rules for primer design (Rybicki)

2.	Degenerate Primers, description (Rybicki)

3.	Incorporation of Restriction Sites, description (Rybicki)

4.	Primer Length, description (Rybicki)


   PCR

Dealing with products

1.	Cleaning PCR Products, protocol (Rybicki)

2.	Detecting Products, description (Rybicki)

3.	Labelling with Digoxigenin, description (Rybicki)

4.	T-A Cloning Strategy, description (Rybicki)


   PCR

Sequencing PCR Products

1.	Sequencing PCR Products, description (Rybicki)


   PCR

Miscellaneous

1.	rePCR unique bands from products of mixed size (Rybicki)
introduction
protocol

2.	summary notes, (Rybicki)


   PCR

Sample reaction

1.	Sample reaction


 PCR, Denaturing temperature and time

Related

Contributor:

Rybicki, E

Reference:

The specific complementary association due to hydrogen bonding of single-stranded nucleic acids is referred to as "annealing": two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" molecule.  One may make nucleic acid (NA) single-stranded for the purpose of annealing -if it is not single-stranded already, like most RNA viruses - by heating it to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling it: this ensures the "denatured" or separated strands do not re-anneal. Additionally, if the NA is heated in buffers of ionic strength lower than 150mM NaCl, the melting temperature is generally less than 100°C - which is why PCR works with denaturing temperatures of 91-97°C.

Taq polymerase is given as having a half-life of 30 min at 95°C, which is partly why one should not do more than about 30 amplification cycles: however, it is possible to reduce the denaturation temperature after about 10 rounds of amplification, as the mean length of target DNA is decreased: for templates of 300bp or less, denaturation temperature may be reduced to as low as 88°C for 50% (G+C) templates (Yap and McGee, 1991), which means one may do as many as 40 cycles without much decrease in enzyme efficiency.

"Time at temperature" is the main reason for denaturation / loss of activity of Taq: thus, if one reduces this, one will increase the number of cycles that are possible, whether the temperature is reduced or not.  Normally the denaturation time is 1 min at 94°C: it is possible, for short template sequences, to reduce this to 30 sec or less.  Increase in denaturation temperature and decrease in time may also work: Innis and Gelfand (1990) recommend 96°C for 15 sec.


 PCR, Annealing Temperature and Time

Related

Contributor:

Rybicki, E

Reference:

Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature (Tm) of a NA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is

Tm = 4(G + C) + 2(A + T)°C.

Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). One should aim at using an annealing temperature (Ta) about 5°C below the lowest Tm of ther pair of primers to be used (Innis and Gelfand, 1990).  A more rigorous treatment of Ta is given by Rychlik et al. (1990): they maintain that if the Ta is increased by 1°C every other cycle, specificity of amplification and yield of products <1kb in length are both increased.  One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches may be tolerated: this is fine if one wishes to amplify similar or related targets; however, it can lead to "non-specific" amplification and consequent reduction in yield of the desired product, if the 3'-most base is paired with a target.  A consequence of too high a Ta is that too little product will be made, as the likelihood of primer annealing is reduced; another and important consideration is that a pair of primers with very different Tas may never give appreciable yields of a unique product, and may also result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand.

Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long.


 PCR, Elongation temperature and time

Related

Contributor:

Rybicki, E

Reference:

This is standardly 70 - 72°C, for 0.5 - 3 min.  Taq actually has a specific activity at 37°C which is very close to that of the Klenow fragment of E coli DNA polymerase I, which accounts for the apparent paradox which results when one tries to understand how primers which anneal at an optimum temperature can then be elongated at a considerably higher temperature - the answer is that elongation occurs from the moment of annealing, even if this is transient, which results in considerably greater stability.  At around 70°C the activity is optimal, and primer extension occurs at up to 100 bases/sec.  About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990).  Longer products require longer times: 3 min is a good bet for 3kb and longer products.  Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting.


 PCR, Reaction buffer

Related

Contributor:

Rybicki, E

Reference:

Recommended buffers generally contain 10-50mM Tris-HCl pH 8.3, up to 50mM KCl, 1.5mM or higher MgCl2, primers 0.2 - 1uM each primer, 50 - 200uM each dNTP, gelatin or BSA to 100ug/ml, and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 (0.05 - 0.10% v/v) (Innis and Gelfand, 1990).  PCR is supposed to work well in reverse transcriptase buffer, and vice-versa, meaning 1-tube protocols (with cDNA synthesis and subsequent PCR) are possible (Krawetz et al., 19xx; Fuqua et al., 1990).  Higher than 50mM KCl or NaCl inhibits Taq, but some is necessary to facilitate primer annealing.  [Mg2+] affects primer annealing; Tm of template, product and primer-template associations; product specificity; enzyme activity and fidelity.  Taq requires free Mg2+, so allowances should be made for dNTPs, primers and template, all of which chelate and sequester the cation; of these, dNTPs are the most concentrated, so [Mg2+] should be 0.5 - 2.5mM greater than [dNTP].  A titration should be performed with varying [Mg2+] with all new template-primer combinations, as these can differ markedly in their requiirements, even under the same conditions of concentrations and cycling times/temperatures.  Some enzymes do not need added protein, others are dependent on it.  Some enzymes work markedly better in the presence of detergent, probably because it prevents the natural tendency of the enzyme to aggregate.

Primer concentrations should not go above 1uM unless there is a high degree of degeneracy; 0.2uM is sufficient for homologous primers.  Nucleotide concentration need not be above 50uM each: long products may require more, however.

Labelling of PCR products with digoxygenin-11-dUTP (DIG; Boehringer-Mannheim) need be done only in 50uM each dNTP, with the dTTP substituted to 35% with DIG-11-dUTP. NOTE: that the product will have a higher MW than the native product!


 PCR, Helix Destabilisers / Additives

Related

Contributor:

Rybicki, E

Reference:

With NAs of high (G+C) content, it may be necessary to use harsher denaturation conditions.  For example, one may incorporate up to 10% (w or v/v) dimethyl sulphoxide (DMSO), dimethyl formamide (DMF), urea or formamide in the reaction mix: these additives are presumed to lower the Tm of the target NA, although DMSO at 10% and higher is known to decrease the activity of Taq by up to 50% (Innis and Gelfand, 1990; Gelfand and White, 1990). Additives may also be necessary in the amplification of long target sequences: DMSO often helps in amplifying products of >1kb.  Formamide can apparently dramatically improve the specificity of PCR (Sarkar et al., 1990), while glycerol improves the amplification of high (G+C) templates (Smith et al., 1990).  It is possible that polyethylene glycol may be a cheap effective additive for increasing reaction rate, by "solvent exclusion", in cases where template concentration is low.


 PCR, Cycle number

Related

Contributor:

Rybicki, E

Reference:

The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting concentration of the target DNA: Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50 target molecules, and 25 - 30 to amplify 3x105 molecules to the same concentration.  This non-proportionality is due to a so-called plateau effect, which is the attenuation in the exponential rate of product accumulation in late stages of a PCR, when product reaches 0.3 - 1.0 nM.  This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs - former a problem with short products, latter for long products); end-product inhibtion (pyrophosphate formation); competition for reactants by non-specific products; competition for primer binding by re-annealing of concentrated (10nM) product (Innis and Gelfand, 1990).

If desired product is not made in 30 cycles, take a small sample (1ul) of the amplified mix and re-amplify 20-30x in a new reaction mix rather than extending the run to more cycles: in some cases where template concentration is limiting, this can give good product where extension of cycling to 40x or more does not.


 PCR, Recommended Reagent Concentrations

Related

Contributor:

Rybicki, E

Reference:

Primers: 0.2 - 1.0 uM

Nucleotides: 50 - 200 uM EACH dNTP

Dimethyl sulphoxide (DMSO): 0 - 10% (v/v)

Taq polymerase: 0.5 - 1.0 Units/50ul rxn

Target DNA: 1 ng - 1 ug (NB: higher concn for total genomic DNA; lower for plasmid / purified DNA / virus DNA target)

Buffer: use proprietary or home-made 10x rxn mix; eg: Cetus, Promega.  This should contain: minimum of 1.5mM Mg2+, usually some detergent, perhaps some gelatin or BSA.  Promega now supply 25mM MgCl2, to allow user-specified [Mg2+] for reaction optimisation with different combinations of primers and targets.

MAKE POOLED MASTER MIX OF REAGENTS IN ABSENCE OF DNA using DNA-free pipette, then dispense to individual tubes (using DNA-free pipette), and add DNA to individual reactions USING PLUGGED TIPS.

OVERLAY REACTIONS WITH 50UL OF HIGH-QUALITY LIQUID PARAFFIN OR MINERAL OIL to ensure no evaporation occurs: this changes reactant concentrations.   NOTE: latest wisdom has it one can use VASELINE - this also allows "HOT START" PCR.

NOTE:

USE PLUGGED PIPETTE TIPS: prevents aerosol contamination of pipettes.

Use of detergents is recommended only for Taq from Promega (up to 0.1% v/v, Triton X-100 or Tween-20). DMSO apparently allows better denaturation of longer target sequences (>1kb) and more product.

DO NOT USE SAME PIPETTE FOR DISPENSING NUCLEIC ACIDS AS YOU USE FOR DISPENSING REAGENTS

Remember sample volume should not exceed 1/10th reaction volume, and sample DNA/NTP/primer concentrations should not be too high as otherwise all available Mg2+ is chelated out of solution and enzyme reactivity is adversely affected.  Any increase in dNTPs over 200uM means [Mg2+] should be re-optimised.

AVOID USING EDTA-CONTAINING BUFFERS AS EDTA CHELATES Mg2+

Low primer, target, Taq, and nucleotide concentrations are to be favoured as these generally ensure cleaner product and lower background, perhaps at the cost of detection sensitivity.


 PCR, Initial Conditions

Related

Contributor:

Rybicki, E

Reference:

Initial denaturation at start:  92 - 97°C for 3 - 5 min. If you denature at 97°C, denature sample only; add rest of mix after reaction colls to annealing temperature (prevents premature denaturation of enzyme).

Initial annealing temperature:  as high as feasible for 3 min (eg: 50 - 75°C).  Stringent initial conditions mean less non-specific product, especially when amplifying from eukaryotic genomic DNA.

Initial elongation temperature:  72°C for 3 - 5 min. This allows complete elongation of product on rare templates.


 PCR, Temperature Cycling

Related

Contributor:

Rybicki, E

Reference:

example conditions

92 - 94°C for 30 - 60 sec (denature)

37 - 72°C for 30 - 60 sec (anneal)

72°C for 30 - 60 sec (elongate) (60 sec per kb target sequence length)

25 - 35 cycles only (otherwise enzyme decay causes artifacts)

72°C for 5 min at end to allow complete elongation of all product DNA

NOTE:

"Quickie" PCR is quite feasible: eg, [94°C 30 sec / 45°C 30 sec / 72°C 30 sec] x 30, for short products (200 -500 bp).

USE GLYCEROL IN THERMAL CYCLER REACTION TUBE HOLES TO ENSURE GOOD THERMAL CONTACTS

DON'T RUN TOO MANY CYCLES: if you don't see a band with 30 cycles you probably won't after 40; rather take an aliquot from the reaction mix and re-PCR with fresh reagents.


 PCR, "Hot Start" PCR

Related

Contributor:

Rybicki, E

Reference:

In certain circumstances one wishes to avoid mixing primers and target DNA at low temperatures in the presence of Taq polymerase: Taq pol is almost as efficient as Klenow pol at 37°C; consequently, if primers mis-anneal at low temperature prior to initial template denaturation, "non-specific" amplification may occur.  This may be avoided by only adding enzyme after the initial denaturation, before the reaction cools to the chosen annealing temperature.  This is most conveniently done by putting wax "gems"TM into the reaction tube after addition of everything except enzyme, then putting enzyme on top of the gem: the wax melts when the temperature reaches +/-80°C, and the enzyme mixes with the rest of the reaction mix while the molten wax floats on top and seals the mix, taking the place of mineral oil.  Latest information is that "gems" may be substituted by VaselineTM.


 PCR, Primer Length

Related

Contributor:

Rybicki, E

Reference:

The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner if one runs the risk of having problems such as described above.  Apart from the Tm, a prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low.  For example, there is a 1/4 chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence.  Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli.  Thus, the association of a greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process, far more so than the specificity of mon°Clonal antibodies in binding to specific antigenic determinants.  Consequently, 17-mer or longer primers are routinely used for amplification from genomic DNA of animals and plants.  Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific priming.


 PCR, Degenerate Primers

Related

Contributor:

Rybicki, E

Reference:

For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences.  For example, Compton (1990) describes using 14-mer primer sets with 4 and 5 degeneracies as forward and reverse primers, respectively, for  the amplification of glycoprotein B (gB) from related herpesviruses.  The reverse primer sequence was as follows:

TC GAATTC NCCYAAYTGNCCNT

where Y = T + C, and N = A + G + C + T, and the 8-base 5'-terminal extension comprises a EcoRI site (underlined) and flanking spacer to ensure the restriction enzyme can cut the product (the New England Biolabs catalogue gives a good list of which enzymes require how long a flanking sequence in order to cut stub ends).

Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases; also, increased degeneracy means concentration of the individual primers decreases; thus, greater than 512-fold degeneracy should be avoided. However, I have used primers with as high as 256- and 1024-fold degeneracy for the successful amplification and subsequent direct sequencing of a wide range of monogeminiviruses against a background of maize genomic DNA (Rybicki and Hughes, 1990).

Some groups use deoxyinosine (dI) at degenerate positions rather than use mixed oligos: this base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present.  This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo.


 PCR, simple set of rules for primer design

Related

Contributor:

Rybicki, E

Reference:

A simple set of rules for primer sequence design

(adapted from Innis and Gelfand, 1991):

1.	primers should be 17-28 bases in length;

2.	base composition should be 50-60% (G+C);

3.	primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;

4.	Tm's between 55-80°C are preferred;

5.	3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;

6.	primer self-complementarity (ability to form 2° structures such as hairpins) should be avoided;

7.	runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.


 PCR, Restriction Sites in Primers

Related

Contributor:

Rybicki, E

Reference:

Incorporation of Restriction Sites in Primers

Although this may be rendered simple by incorporating the same or different restriction sites at the 5'-ends of PCR primers - which allows generation of sticky ends and straightforward cloning into appropriate vectors -these should have AT LEAST two additional bases 5' to the recognition sequence to ensure that the enzymes will in fact recognise the sequence - and it is often found that even when this is done, the efficiency of cutting of fresh product is next to zero.  This can sometimes be remedied by incubating fresh product with Proteinase K (to digest off tightly-attached Taq pol), but often is not.  A solution to the problem is to use the "Klenow-Kinase-Ligase" (KKL) method: this involves "polishing" products with Klenow, kinasing them to get 5'-phosphorylation (NB: OLIGONUCLEOTIDE PRIMERS NORMALLY HAVE NO 5'-PHOSPHATES!!!), ligating the fragments together to get concatemers, then restricting these with the appropriate restriction enzymes to generate the sticky-ended fragments suitable for cloning (Lorens, 1991; PCR Methods and Applications, 1: 140-141).


 PCR, Summary

Related

Contributor:

Rybicki, E

Reference:

AND ALWAYS REMEMBER:

- WORK CLEAN

- TITRATE MAGNESIUM

- DON'T USE TOO MUCH TEMPLATE DNA

- DON'T USE PCR PRODUCTS IN PCR PREPARATION

   	  AREAS

- ALWAYS, ALWAYS INCLUDE WATER AND VERY DILUTE

  POSITIVE CONTROLS IN EVERY EXPERIMENT

- WEAR GLOVES

- USE PLUGGED TIPS


"CORE SAMPLE" PCR


 PCR, Asymmetric PCR

Related

Contributor:

Rybicki, E

Reference:

Asymmetric PCR for ssDNA Production

Simply use a 100:1 molar ratio of the two primers (eg: primer 1 at 0.5uM, primer 2 at 0.005uM).  This allows production of  mainly ssDNA of the sense of the more abundant primer, which is useful for sequencing purposes or making ssDNA probes.


 PCR, cDNA PCR

Related

Contributor:

Rybicki, E

Reference:

cDNA PCR

A very useful primer for cDNA synthesis and cDNA PCR comes from a sequencing strategy described by Thweatt et al. (1990): this utilised a mixture of three 21-mer primers consisting of 20 T residues with 3'-terminal A, G or C, respectively, to sequence inside the poly(A) region of cDNA clones of mRNA from eukaryotic origin.  I have used it to amplify discrete bands from a variety of poly(A)+ virus RNAs, with only a single specific degenerate primer upstream: the T-primer may anneal anywhere in the poly(A) region, but only molecules which anneal at the beginning of the poly(A) tail, and whose 3'-most base is complementary to the base next to the beginning of the tail, will be extended.


 PCR, Detecting Products

Related

Contributor:

Rybicki, E

Reference:

Take 1/10th - 1/3rd of the reaction mix CAREFULLY from under the oil or from under the Vaseline or solidified wax, using a micropipette with plugged tip, IN AN AREA AWAY FROM YOUR PCR PREPARATION AREA!  Mix this with some gel loading buffer(1:1 - 1:5 mix:loading buffer): this is TBE containing 10 - 20% glycerol or sucrose and a dash of bromophenol blue (BPB) tracking dye.  Load 5 -30ul of sample into wells of 0.8 - 3.0% submarine agarose gel made up in TBE, preferably containing 50ng/ml ethidium bromide.  Run at 80 -120 volts (not too slow or small products diffuse; not too fast or bands smear) until BPB reaches end of gel (large products) or 2/3 down gel (small products).  Use DNA markers going from 2kb down to 100 bp or less (recommend BM PCR markers).  View on UV lightbox at 254 -300 nm, photo 1 -5 sec.

NOTE:

Small products are best seen on 3% agarose gels that have been run fast (eg: 100 volts), with BPB run to 1/2 - 2/3 down the gel.  It is best to include EthBr in the gel AND in the gel buffer, as post-electrophoresis staining can result in band smearing due to diffusion, and if there is no EthBr in the buffer the dye runs backwards out of the gel, and smaller bands are stripped of dye and are not visible.

NUSIEVETM gel (FMC Corp) can also be used for small products - better resolution than agarose.

Polyacrylamide gels can be silver stained by simple protocols for extreme sensitivity of detection.

Gels can be blotted directly after soaking in 0.5M NaOH / 1.5M NaCl for 10-20 min: "dry blotting" works well (eg: gel is over- and under-layered with paper towel stacks and pressed; bands transfer up and down), as does classic "Southern" blotting.  Bands blotted in this way are already covalently fixed onto nylon membranes, and simply need a rinse in 5xSSPE before prehybridisation.


 PCR, Labelling with Digoxigenin

Related

Contributor:

Rybicki, E

Reference:

PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790).  This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions.  It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.

 	Make a DIG-dNTP mix for PCR as follows:

DIG NUCLEOTIDE MIX	CONCENTRATIONS

Dig-11-dUTP	350 uM

dTTP	650 uM

dATP	  1 mM

dCTP	  1 mM

dGTP	  1 mM

For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above.  The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically.  Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured.  Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature.


 PCR, Cleaning PCR Products

Related

Contributor:

Rybicki, E

Reference:

1)	Getting rid of mineral oil: simply add 50ul of chloroform to the reaction vial, vortex and centrifuge briefly, and remove the "hanging droplet" of AQUEOUS solution with a micopipette.

2)	Getting rid of wax or Vaseline: simply "spear" wax gem and remove; do as for oil or bottom-puncture tube and blow out aqueous drop for Vaseline.

3)	Cleaning-up DNA: 3 options:

a)	a protocol which gives DNA that is clean enough for sequencing is the following: increase volume to 100ul with water, add 10M ammonium acetate soln. to 0.2M final concentration (1/5th volume), add equal volume of isopropanol (propan-2-ol), leave on bench 5 min, centrifuge 20 min at 15 000 rpm, remove liquid using pipette, resuspend in 100ul water or TE, repeat precipitation.

b)	Simply do a phenol-CHCl3 extraction (add 20ul phenol to aqueous phase, vortex, add 50ul CHCl3, vortex, centrifuge, remove UPPER aqueous phase, repeat CHCl3 extraction).

c)	Make aqueous phase up to 400ul, and spin through Millipore Ultrafree-MC NMWL 30 000 cartridges (at 6000 rpm in micr°Centrifuge), wash through with 2x400ul water, collect +/-20ul sample: this is pure enough for sequencing.

NOTE:

Product is clean enough for restriction digest immediately after reaction; however, phenol-chloroform clean-up is recommended as a minimum.


 PCR, T-A Cloning Strategy

Related

Contributor:

Rybicki, E

Reference:

Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated addition of a single nucleotide to the 3'-ends of PCR products.  In the presence of all 4 dNTPs, dA is preferentially added; however, use of a single dNTP in a reaction mix results in (relatively inefficient) addition of that nucleotide.  This complicates cloning, as the supposedly blunt-ended PCR product often is not, and blunt-ended cloning protocols often do not work or are very inefficient.  This can be remedied by incubation of PCR products with T4 DNA pol or Klenow pol, which "polishes" the ends due to a 3'->5' exonuclease activity (Lui and Schwartz, 1992; BioTechniques, 20: 28-30).  However, this terminal transferase activity is also the basis of a clever cloning strategy: this uses Taq pol to add a single dT to the 3'-ends of a blunt-cut cloning vector such as pUC or pBluescriptTM, and simple ligation of the PCR product into the now "sticky-ended" plasmid (Marchuk et al., 1990; NAR 19: 1156).


 PCR, Sequencing PCR Products

Related

Contributor:

Rybicki, E

Reference:

This is best done using ssDNA generated by asymmetric PCR, and the "limiting" primer for sequencing.  However, efficient sequencing of dsDNA generated by normal PCR is possible using  the modification to the SequenaseTM protocol published by Bachmann et al. (1990) (NAR 18: 1309).  CLEAN DNA is resuspended in sequencing buffer containing 0.5% Nonidet P-40 and 0.5% Tween-20 and sequencing primer, denatured by heating to 95°C for 5 min, snap-cooled on wet ice, and sequenced by the "close-to-primer" protocol (eg: dilute extension mixes).


 PCR, Introduction to re-PCR

Related

Contributor:

Rybicki, E

Reference:

a method to rePCR unique bands from products of mixed size

Introduction

The products of a PCR reaction  especially when this is done on eukaryotic genomic DNA, and when using degenerate primers  often contain a mixture of discretesized bands, one of which is the "right" one, while the others represent products of "nonspecific" priming.  It can be a problem to obtain the correct band in any state approaching purity while maintaining yield, and attempting to purify the band by cloning all the reaction products and then probing the library for the correct DNA can be extraordinarily tedious.

I have applied a simple "core sampling" procedure involving "coring" an agarose sample out of a gel, and using it as template for another round of PCR  to get around this problem, and obtain unique bands from initially messy backgrounds.  Of course, having a visible band of the size expected does help; however, the technique may be used on faith on "rightsized" invisible bands if need be.

Comments

I have successfully reamplified a unique 500bp band from a background of many bands up to 1.5kb from a cDNA PCR of cauliflower mosaic virus 35S RNA in total turnip RNA extract, and a 150bp band from a background of bands going up to 3kb from an amplification of Arabidopsis total genomic DNA using thoroughly degenerate primers  in the latter case, to a point where it could be sequenced directly (using same primers) after a subsequent amplification after purification from a gel plug as above.

The method has advantages over a previouslydescribed toothpicking procedure in that a core sample is generally of defined volume, may be stored indefinitely, and provides material for multiple reamplifications.


 PCR, protocol for re-PCR

Related

Contributor:

Rybicki, E

Reference:

a method to rePCR unique bands from products of mixed size

Protocol

1.	Run products of a PCR amplification on 12% TBE agarose gel, as two or more replicate lanes.

2.	Cut off 1 lane  flanked by marker DNA if desired, and notched to allow reorientation with remainder of gel and stain in preferred ethidium bromide concentration (I use 50 ng/ml for 10 min).

3.	View excised stained piece on 254nm UV box for maximum senssitivity; notch or stab correct band(s) in sample lane.

4.	Prepare "core samplers": using gloves and sterile scissors and cut off about 5mm from the tip of as many sterile yellow pipette tips (we use Gilson tips) as you will need for samples.

5.	Align stained marked segment with remainder of gel.  Use "core samplers" to stab out one or more cores of agarose from the centre of bands of interest, using stabbed/notched gel lane as reference: a standard gel should give about 10ul per core.

6.	Stain remainder of gel, view and photograph at 254nm to ensure correct regions were sampled.

NOTE: IT IS POSSIBLE TO QUICKLY CORE A STAINED GEL DIRECTLY ON A 305 OR EVEN A 254 NM UV BOX; HOWEVER, MORE THAN A FEW SECONDS OF EXPOSURE RESULTS IN CROSSLINKING AND NO AMPLIFICATION

7.	Use core samples as substrate in PCR reactions: I make up 40ul/reaction of reaction mix, and allow 10ul per core.  Simply add core to mix, vortex a little, spin down, cover with mineral oil.  PCR according to taste (not inhibited by presence of a little bromophenol blue or of 50ng/ml ethidium bromide).

8.	At end of PCR: if you allow the tubes to cool down the reaction mix will set: 2%odd agarose diluted 1/5 sets quite well!  This is no problem for gel running as you then end the PCR on a 10 min 72°C cycle, and load the sample into wells of a gel BEFORE submeging the gel: sample will set in the wells and not float out.

9.	If you wish to extract DNA, end at 72°C and add 50ul prewarmed phenol / 8OHquinoline and vortex, add 100ul chloroform / isoamyl alcohol (24:1), vortex, spin: agarose should be in the phenol/CHCl3 phase. ALTERNATIVELY: take off mineral oil using 50ul CHCl3, take out plug of solidified sample and wash in TE, then put into 0.5ml Eppendorftype vial with some siliconised glass wool at bottom, and a small needle hole.  Put little Eppi in big Eppi without a lid, and spin 6000 rpm 10 min (a la Heery et al., 1990; TIG 6(6):173).  Collect filtrate, clean up by phenol/CHCl3 and isopropanol/ammonium acetate ppte (1 vol IP, 0.2 vol of 10M ammonium acetate).


 PCR, References

Related

Contributor:

Rybicki, E

Reference:

Compton T (1990).  Degenerate primers for DNA amplification.pp. 3945 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Fuqua SAW, Fitzgerald SD and McGuire WL (1990).  A simple polymerase chain reaction method for detection and cloning of lowabundance transcripts.  BioTechniques 9 (2):206211.

Gelfand DH and White TJ (1990).  Thermostable DNA polymerases.  pp. 129141 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Innis MA and Gelfand DH (1990).  Optimization of PCRs.  pp. 312 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Krawetz SA, Pon RT and Dixon GH (1989).  Increased efficiency of the Taq polymerase catalysed polymerase chain reaction.  Nucleic Acids Research 17 (2):819.

Rybicki EP and Hughes FL (1990).  Detection and typing of maize streak virus and other distantly related geminiviruses of  grasses by polymerase chain reaction amplification of a conserved viral sequence.  Journal of General Virology 71:25192526.

Rychlik W, Spencer WJ and Rhoads RE (1990).  Optimization of the annealing temperature for DNA amplification in vitro.  Nucleic Acids Research 18 (21):64096412.

Sarkar G, Kapeiner S and Sommer SS (1990).  Formaqmide can drastically increase the specificity of PCR.  Nucleic Acids Research 18 (24):7465.

Smith KT, Long CM, Bowman B and Manos MM (1990).  Using cosolvents to enhance PCR amplification.  Amplifications 9/90 (5):1617.

Thweatt R, Goldstein S and Reis RJS (1990).  A universal primer mixture for sequence determination at the 3' ends of cDNAs.  Analytical Biochemistry 190:314316.

Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991).  The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.  DNA and Cell Biology 10 (3):233238.

Yap EPH and McGee JO'D (1991).  Short PCR product yields improved by lower denaturation temperatures.  Nucleic Acids Research 19 (7):1713.


  DNA methods

Sequencing

1.	Pharmacia

2.	Pharmacia - GC rich

3.	Promega taqtrack deaza sequencing

4.	Extra long gels

5.	Gel preparation

6.	Gel pouring

7.	setting up sequencing apparatus

8.	notes on sequencing techniques


Sequencing protocols, description
  Sequencing protocols

Related

Contributor:

James, Di

Reference:

various

Various sequencing protocols are available for the dideoxy method.

1.	Pharmacia

2.	Pharmacia - GC rich

3.	Promega taqtrack deaza sequencing

4.	Extra long gels

5.	Gel preparation

6.	Gel pouring

7.	setting up sequencing apparatus

8.	notes on sequencing techniques


Sequencing protocols, pharmacia, description
  Sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

DNA sequencing  pharmacia protocol
Alkaline denaturation of ds template
Priming reaction
Termination tubes
Labelling reaction
Termination reaction

DNA sequencing  pharmacia gcrich protocol
Alkaline denaturation of ds template
Priming reaction
Termination tubes
Labelling reaction
Termination reaction


Sequencing protocols, pharmacia, denaturation
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Alkaline denaturation of ds template

1.	10 mg DNA (1 pMol) Maxiprep (NUCLEOBOND or CsCl)

2.	Dilute to 18 ul vol with Sterile deionised distilled water

3.	Add 2 ul of of 2 M NaOH incubate at 37°C for 30 min

4.	Place on ice, immed. add 3 ul of 3 M Na acetate plus 100 ul of 96% EtOH.

5.	Place @ 70°C for 515 min.  Spin for 20 min @ 4°C

6.	Wash pellet with 500 ul 70% EtOH.  Change orientation.

Spin 10 min @ 4°C, remove all supernatant carefully and (almost) dry

Media, buffers and solutions

sodium acetate

sodium hydroxide


Sequencing protocols, pharmacia, priming
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Priming reaction

To dried denat. template add 10 ul of primer/buffer/water mix

Number  of  templates

Component	 1	 2	 3	 4

μl Primer (2 pmol)	 2	 4	 6	 8

μl Annealing Buffer	 2	 4	 6	 8

μl Deionised Dist.Water	10	20	30	40

_________________________________________________________

1.	Incubate @ 37°C(AT) for 30'.  Mix occasionally to resuspend pellet.

2.	Place tubes in container of water @ 37°C and slowly cool to RT ~ 30 min.

Media, buffers and solutions


Sequencing protocols, pharmacia, termination tubes
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Termination tubes

Label 4 tubes per template:  A (green), C (red), G (blue), T (orange)

________________________________________________________

ddNTP (long or short)	 1	 2	 3	 4

________________________________________________________

μl Termination mix (+ Ext. mix dNTP)	2.50	6.30	8.80	10.0

μl DMSO	0.25	0.63	0.88	 1.0

_________________________________________________________

Use 2.75 μl of above.  Mix to appropriate Termination Mix Tube.

Media, buffers and solutions


Sequencing protocols, pharmacia, labelling
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Labelling reaction

Keep tube on ice:  T7 Polymerase @ 20°C.

________________________________________________________

Component	1	2	3	 4

_________________________________________________________

μl DMSO	1.5	3.0	4.5	 6.0

μl Lab Mix dATP (or dCTP)	3.0	6.0	9.0	12.0

μl Enz. Dil. Buffer + glycerol	1.58	3.15	4.74	 6.32

μl T7 Polymerase	0.42	0.85	1.26	 1.68

μl Labelled dATP (35S) (or dCTP)	0.5	1.0	1.5	 2.0

__________________________________________________________

Place template tubes at room temperature.

Add 6.0 ul  of above mix to each template and incubate for 4 - 10 min.

Media, buffers and solutions


Sequencing protocols, pharmacia, termination reaction
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Termination reaction

1.	Prewarm the four (A,C,G,T) Termination tubes to 37°C for 30 sec.

2.	Aliquot 4.5 ul from template @ RT into each of the 4 prewarmed termination tubes.

3.	Incubate for exactly 5 min @ 37°C.

4.	Add 5 ul of Stop Buffer to each tube and store on ice or @ 20°C.

5.	Denature templates by heating for 2 min @ 8595°C.  Spin & place on ice stat.

6.	Load 3 ul for long and medium runs:  4 ul for short runs.

7.	Set volts or watts constant to give 42 mAmps at start (will stabilize @ +42°C

8.	Short = 2 h.  Medium = 4 h.  Long = 6 h.    (if far from vector)
           1.5 h              3.5 h           5.5 h     (if close to vector)

Run gels in Taurine buffer.

Media, buffers and solutions

Taurine buffer


Sequencing protocols, pharmacia, priming (GC rich)
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Priming reaction for GC rich templates

  To dried denat. template add 10 ul of primer/buffer/water mix

________________________________________________________

Number  of  templates	 1	 2	 3	 4

_________________________________________________________

μl Primer (2 pmol)	 2	 4	 6	 8

μl Annealing Buffer	 2	 4	 6	 8

μl Deionised Dist.Water	10	20	30	40

_________________________________________________________

Incubate @ 65°C(GC) for 5' or 37°C(AT) for 30'.

Mix occasionally to resuspend pellet.

Place tubes at -20°C or use immediately.

Media, buffers and solutions


Sequencing protocols, pharmacia, termination tubes (GC rich)
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Termination tubes for GC rich templates

Label 4 tubes per template:  A (green), C (red), G (blue), T (orange)

________________________________________________________

ddNTP (long or short)	1	2	3	 4

________________________________________________________

ul Termination mix + (dNTP ext. mix)	2.50	6.30	8.80	10.0

ul DMSO	0.25	0.63	0.88	 1.0

__________________________________________________________

Use 2.75 ul of above.

Mix to appropriate Termination Mix Tube.

Media, buffers and solutions


Sequencing protocols, pharmacia, labelling (GC rich)
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Labelling reaction for GC rich templates

Keep tube on ice:  T7 Polymerase @ 20°C.

__________________________________________________________________________

Component	1	2	3	 4

__________________________________________________________________________

μl DMSO	1.5	3.0	4.5	 6.0

μl Lab Mix dCTP	3.0	6.0	9.0	12.0

μl Enz. Dil. Buffer with glycerol	1.58	3.15	4.74	 6.32

μl T7 Polymerase	0.42	0.85	1.26	 1.68

μl Labelled dCTP (35S)	0.5	1.0	1.5	 2.0

μl Pyrophosphatase buffer	0.125	0.25	0.38	 0.5

__________________________________________________________________________

Place template tubes at room temperature.

Add 6.0 ul  of above mix to each template and incubate for 4 - 10 min.

Media, buffers and solutions


Sequencing protocols, pharmacia, termination (GC rich)
  Pharmacia Sequencing protocol

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Termination reaction for GC rich templates

1.	Prewarm the four (A,C,G,T) Termination tubes to 48°C for 30 sec.

2.	Aliquot 4.5 ul from template @ RT into each of the 4 prewarmed termination tubes.

3.	Incubate for exactly 10 min @ 48°C.

4.	Add 1 ul 0.25 u Klenow mix, incubate 4 min.

5.	Add 5 ul of Stop Buffer to each tube and store on ice or @ 20°C.

6.	Denature templates by heating for 2 min @ 8595°C.  Spin & place on ice stat.

7.	Load 3 ul for long and medium runs:  4 ul for short runs.

8.	Set volts or watts constant to give 42 mAmps at start (will stabilize @ +42°C

9.	Short = 2 h.  Medium = 4 h.  Long = 6 h.    (if far from vector)

          1.5h                3.5 h           5.5 h   (if close to vector)

Run gels in Taurine buffer.

Media, buffers and solutions

Taurine buffer


Sequencing protocols, promega, description
  Sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

DNA sequencing  promega taqtrack deaza protocol
Alkali denaturation
Annealing ds plasmid template and primer
Termination tubes
Extension/labelling reaction
Termination reaction


Sequencing protocols, promega, denaturation protocol
  Promega sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Alkali denaturation of supercoiled plasmid DNA

1)	Add 510mg of supercoiled plasmid DNA to a microcentrifuge tube and add deionised water to a final volume of 18ul.

2)	Add 2ul of 2M NaOH and incubate for 10 minutes at 37°C.

3)	Add 3ul of 3M sodium acetate, pH 7.5.

4)	Add 112ul of 100% ETOH (icecold).

5)	Incubate at 70°C for 5'

6)	Centrifuge at 4°C for 10'.

7)	Decant the supernatant and wash the pellet with 500 ul icecold 70% ETOH

8)	Change orientation, spin 10', Remove the 70% ETOH and almost dry pellet.

Media, buffers and solutions


Sequencing protocols, promega, priming protocol
  Promega sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Annealing doublestranded plasmid template and primer

1)	Add 1ul of 20ng/ul primer
5ul buffer
14.5 ul of dH20 to the pellet
(final volume of 20.5ul)

2)	Incubate at 65°C for 10 mins and continue immediately with labelling

Media, buffers and solutions


Sequencing protocols, promega, termination tubes
  Promega sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Termination tubes

1)	Label 4 tubes for each template A, C, G, and T

2.	aliquot 1ul (with 10% DMSO) of the appropriate d/ddNTP mixture
(usually ratio 1.5x ddNTPs to 1.0x dNTPs EXT MIX) into each tube.

Media, buffers and solutions


Sequencing protocols, promega, labelling protocol
  Promega sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Extension/labeling reaction

1)	Mix a cocktail containing the following ratio of contents:
2.5ul DMSO
2ul ext/lab mix (dATP or dCTP)
0.5ul Taq polymerase
1ul S35 labeled nucleotide

2)	Add 6ul of the cocktail to each primed template.

3)	Incubate at 70°C for 5'.

Media, buffers and solutions


Sequencing protocols, promega, termination protocol
  Promega sequencing protocols

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Termination reaction

1)	Aliquot 6.4ul of labeled template to each of the appropiate ddNTP tube

2)	Incubate the tubes at 70°C for 15 minutes.

3)	Add 4ul of stop solution to each tube and store at 20°C.

4)	Heat the tubes to > 85°C for 25 minutes then immediately place on ice/water mix, before loading.(2ul per well, for shorts/meds/longs)

Media, buffers and solutions


Sequencing gel preparation
  Gel preparation

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

1.	Mark the outer surface of the plates with tape.  Plates should be held by the edges  the inner surface should never be touched.

2.	Wash the inner sides with a sponge and CHEMICO LIQUICLEEN or HANDY ANDY BLUE.  Rinse off all traces of detergent using paper towelling and water.  Place on bench and rinse with D. water  dry  rinse with EtOH  dry.

3.	Place plate with EARS on bench, add a few good drops of CThru and GENTLY using paper towelling brush up and down along the entire surface.

Leave to dry for 10 minutes.

Wash gently with EtOH.

4.	Wet spacers with D. water lay shortest one on bottom edge of plate with no ears.  Lay longer spacers on sides of plate.  Take great care that where spacers join they do not overlap.

Place plate with ears on top of plate with spacers.

Rearrange spacers making sure there is no overlap or gaps.

Clamp. Use small clamp 10 cm from top to facilitate the insertion of another large spacer to form interface.  Place plates on gel pouring apparatus.

Materials

2 plates	40x30 cm (1 with short flat ears)

3 spacers	2 of 1x42 cm and 1 of 1x32 cm

clamps	5 large, 2 medium 9 small


  sequencing gel pouring

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Have ready:

a)	50 ml syringe without plunger but with 0.8 μm filter.

b)	2 clean 100 ml beakers

1.	To one beaker add 50 ml 5% gel mix into the 50 ml syringe

a.	Filter and add 45 ul 50% Am. Pers + 45 ul TEMED.

b.	Pour from one beaker to the other slowly and back again

c.	Draw up 50 ml into syringe.

d.	With smooth jerkless action pour gel mix down one side of prepared plates and slowly lift horizontally as plates fill.

e.	Insert flat edge of large spacer 3  4 mm into top of plate.

f.	Place 3 large clamps along top of plates to compress large spacer.

g.	Cover with cling wrap if plates are only going to be used next day.

h.	Polymerisation should occur within 20 - 30 min

Materials, buffers and solutions

50% Amonium persulphate must be made up fresh each week (0.25 g + 500 ml distilled water)

TEMED

gel mix


  sequencing apparatus setup

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

1.	Warm 2 litres 1X TBE or TTE in microwave on high 6 mins.

2.	Place plates on polystyrene block and plate with ears on top.

3.	Remove glad wrap and all clamps from the glass plates.

4.	Remove BOTTOM SPACER and TOP SPACER ONLY:  DO NOT REMOVE SIDE SPACER.

5.	Fill bottom space with 1X TBE or TTE.

6.	Wash out top space well with 1X TBE or TTE and check condition of gel at top.  (sometimes bubbles have formed where the spacer was, these must be marked at not used).

7.	Clean top glass plate, with ears, very well with 1X TBE or TTE and dry.

8.	Check that the seals on apparatus are still slightly greased (high vacuum grease).

9.	Add 1cm of 1X TBE or TTE to bottom tank.

10.	Place plates on apparatus, eared plate facing inward and clamp with 3 or 4 clamps each side, bottom clamp .5cm from bottom tank.

11.	Add warmed 1X TBE or TTE to tap tank filling to 1cm from top.  Check for leaks, if leaking add more clamps to bottom, if still leaking, take apart and repeat, greasing seals with vacuum grease.

12.	No leaks, add 1X TBE or TTE buffer to bottom tank.

13.	Rinse out top of gel 4 times with 1X TBE or TTE using syringe to remove ureafrom meniscus.

14.	Place shark tooth comb (points facing down) between plates until just piercing gel.

15.	Add ± 2ul of stop dye to each alternate well.

16.	Prerun @ 80 watts (± 42 mAmps) for 510mins.

(At the end of 1.5 hours readings should be ± 2500 volts, ± 35 mAmps)

17.	Switch off POWERPACK.

18.	NB: REWASH wells to remove urea before adding templates.

19.	Load 4ul for shorts  :  80 watts 1/12 hrs.

3 ul meds and longs  :  80 watts 3/12 & 6 hrs.

20.	For med and long runs, change bottom tank buffer only every 2 hrs.

21.	When temp reaches 50°C:  Place fan on in front to maintain temp 40  42°C.

22.	Enter run number and template position in book.

23.	On completion:  SWITCH OFF POWERPACK.  Remove bottom tank buffer first using suction tube and then top tank buffer.

24.	Place eared plate upwards and remove comb and side spacers.

25.	Using spatula (with bevelled edge) under left ear. GENTLY EASE plates apart and in one motion lift off top plate.  (Do not EASE spatula under central loading edge).

26.	Label top left hand corner of precut 3mm filter paper and place downwards on gel.  Gently smooth, particularly corner edges.

27.	Lift far edge and check gel has adhered to paper and then roll over hand towards you in one motion.

28.	Place on geldryer grid and cover with glad wrap.

29.	Place on dryer 75  80°C for 3/4 hrs.

30.	Remove glad wrap and place gel in cassettes securing down edges.

31.	Place film on top of gel turning left hand corner of film to mark gel start.

32.	Expose O/N and check the next day.

Materials

TBE

TTE

CAUTION

*********************************************************

* Unpolymerized acrylamide is a potent neurotoxin.      *

* Handle with extreme care, and use gloves to protect   *

* hands.  Dispose of acrylamide contaminated materials  *

* in the special plastic bag lined dustbins found at    *

* the sequencing stations. TEMED is highly toxic.       *

* Store in fridge. RESPIRATOR MASKS MUST BE USED AT     *

* ALL TIMES WHEN HANDLING ACRYLAMIDE, BIS OR TEMED.     *

*********************************************************


Sequencing, notes on sequencing techniques
  notes on sequencing techniques

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

CAUTION

*********************************************************

* Unpolymerized acrylamide is a potent neurotoxin.      *

* Handle with extreme care, and use gloves to protect   *

* hands.  Dispose of acrylamide contaminated materials  *

* in the special plastic bag lined dustbins found at    *

* the sequencing stations. TEMED is highly toxic.       *

* Store in fridge. RESPIRATOR MASKS MUST BE USED AT     *

* ALL TIMES WHEN HANDLING ACRYLAMIDE, BIS OR TEMED.     *

*********************************************************

First read sequenase booklet:

      "Step by step protocols for DNA sequencing"

Respiratory masks must be worn when handling temed and acrylamide

1.	Double stranded DNA of 510 ug amounts; use 10 ng primer, GCrich use 2060 ng.

2.	I routinely use 10 ug of DNA.

3.	For denaturing dsDNA use fresh 2 N NaOH (max 1 week old).

Neutralize at 0°C with either 2 M NH4 acetate or Na acetate.

At end of denaturing step do not overdry pellet.

Continue with priming step immediately or store dried pellet at 20°C and when commencing with priming first add primer before buffer and d.w. (or simultaneously). Primed templates may be stored at 4°C for 48 h or at 20°C for 2 weeks (see note 12 for GCrich).  If template has been stored at 20°C after priming reaction, check that the volume is still 10 ul.  If not, make up to 10 ul, otherwise you will not have enough for termination step.

4.	Priming may either be done at 37°C for 30 min and cooled slowly to RT, or at 100°C or 65°C for 5 min and cooled slowly to RT, or as protocol.

5.	Termination tubes may be aliquotted the previous day and stored with the ext. mix and DMSO at 4°C.

6.	Use colour coding to label termination tubes,

eg. Green = A, Red = C, Blue = G, Brown = T.

7.	Always add reagents for labelling reaction mix in order as per protocol.  Keep T7 polymerase at 20°C, add to Eppendorf in freezer or fridge.  Keep mix on ice throughout labelling.  KEEP ALL REAGENTS ON ICE WHEN THAWED.

8.	Check activity date of 35S and add extra if needed.

NB: dATP or dCTP 35S must be well mixed before use.

9.	LAB MIX is diluted in d.w. and a 1:5 dilution is normally used (USB).

10.	If A&T or G&C rich a 1.5x extension mix should be used for the relevant termination tubes.

11.	Urea masks the 35S signal to a degree.  Gel washing enhances the signal but it is not necessary with our protocol so we do dry lifts and use CURIX RP1 Xray film.

12.	When sequencing GCrich anneal primer, do reactions and run gels on the same day.  Use freshly transformed templates.

13.	Use fresh gel mix (1 week max) for normal runs; short med. long.

14.	Keep TEMED in fridge.

Materials, buffers and solutions

125 ml gel mix

150 ul 50% Amm. Persulphate

100 ul TEMED


Sequencing, extra long gels
  protocol for running extra long gels

Related        Sequencing methods

Contributor:

James, Di

Reference:

?

Caution:

 caution: acrylamide and TEMED

Pouring of gel.

1.	Stand joined plates straight up and pour down one side until 60-80 ml added.  Lie flat and press hard.  Try to add another 100 ml. Insert comb and seal with 5 clamps.

2.	Once the gel has polymerised.  Remove bottom spacer and comb, wash out and fill bottom and top space with 1x Taurine buffer.  Wash and dry outside of top plate, with ears, with DW.

3.	Clamp top tank onto top of plates.

4.	Fill bottom tank top to 1 cm with 1x Taurine buffer.

5.	Place on apparatus with top tank at the back and clamp each side with 3 clamps.

6.	Fill top tank with 1x Taurine buffer.  Check for leaking.  Fill bottom tank with 1x Taurine buffer.

7.	Clean out top space well using syringe.

8.	Carefully insert shark's tooth comb.

9.	Add dye to every second well, check for leaking.

10.	Prerun the gel for 1 hour @ 50 watts (2360 volts 21 Ams to start).

11.	Switch off powerpack.  Rinse out wells thoroughly.

12.	Load samples (1.5  3 ul).  Run @ 50 watts or 2800 volts.

20 hrs = overlap with 2 hrs TBE.  16 hrs = overlap with 11/2 hrs TBE.

13.	Place fan on my bench facing glass plates and leave on O/N.

Place danger sign to warn off others.

Materials, buffers and solutions

125 ml gel mix

150 ul 50% Amm. Persulphate

100 ul TEMED


Recipe for TBE buffer
  TBE (10x)

Contributor:

James, D

Related:

Sequencing

TBE (10x)

Component 	stock conc	final conc.	/ 1l

Tris	108 g

Boric Acid 	 	55 g

EDTA, pH 8.0	0.5M	40 ml

water	to 1l

autoclave


Recipe for TTE (Taurine) buffer
  TTE (10x), Taurine buffer

Contributor:

James, D

Related:

Sequencing

Taurine buffer, TTE (10x)

Component 	stock conc	final conc.	/ 1l

Tris	108 g

Taurine	 	36 g

EDTA	3.72g

water	to 1l


Recipe for acrylamide gel mixes
  Acrylamide gel mixes

Contributor:

James, D

Related:

Sequencing

Acrylamide gel mix (various percentage)

Component 	stock conc	final conc.	4% 100ml	5% 100ml	6% 100ml

Acrylamide (BDH)	 3.8 g	 4.7 g	 5.7 g

Bis (Biorad or Stratagene)	 	0.2 g	 0.25 g	 0.3 g

Urea (Stratagene)	48.0 g	48.0 g	48.0 g

TBE or TAURINE	10x	10.0 ml	10.0 ml	10.0 ml

Dist Water	40.0 ml	40.0 ml	40.0 ml

Mix + dissolve with heat (max. 55°C).  Cool.  Make up to 100 ml.

Dist Water	to 100ml	to 100ml	to 100ml

Acrylamide gel mix (various percentage) for long sequencing gels

Component 	stock conc	final conc.	6% 125ml	6% acryolgel 5 125ml

Acrylamide (BDH)	 7.13g	18.5ml

Bis (Biorad or Stratagene)	 	0.38g	-

Urea (Stratagene)	60g	64.25g

TAURINE (TTE)	10x	12.5ml	12.5ml

Dist Water	50.0 ml	25.0 ml

Mix + dissolve with heat (max. 55°C).  Cool.  Make up to 100 ml.

Dist Water	to 125ml	to 125ml

NB  gel must be poured on the same day it is made and run after 1 hour or overnight.