APPENDIX 2D
Common Buffers, Media, and Stock Solutions
This section describes the preparation of selected bacterial media and of buffers andreagents used in the manipulation of nucleic acids and proteins. Recipes for cell culturemedia and reagents are located in
APPENDIX 3G
and for yeast culture media in Chapter 5.
GENERAL GUIDELINES
When preparing solutions, use deionized, distilled water and (for most applications)reagents of the highest grade available. Sterilization is recommended for most applica-tions and is generally accomplished by autoclaving. Materials with components that arevolatile, altered or damaged by heat, or whose pH or concentration are critical should besterilized by filtration through a 0.22-
µ
m filter. In many cases such components are addedfrom concentrated stocks after the solution has been autoclaved. Where specializedsterilization methods are required, this is indicated in the individual recipes.
CAUTION:
It is important to follow laboratory safety guidelines and heed manufacturers’precautions when working with hazardous chemicals; see
APPENDIX 2A
for further details.
STORAGE
Most simple stock solutions can be stored indefinitely at room temperature if reasonablecare is exercised to keep them sterile; where more rigorous conditions are required, thisis indicated in the individual recipes.
SPECIAL CONSIDERATIONS FOR PCR EXPERIMENTS
Because the polymerase chain reaction is designed to detect very small amounts of DNA,only a few molecules of contaminating DNA will produce unwanted amplificationproducts. Ideally, PCRs should not be carried out in the same room where large quantitiesof DNA are handled. Even where such spatial separation is not practical, the followinghousekeeping procedures will help avoid contamination with extraneous DNA (H.D. Kay,pers. comm.).1. Keep laboratory surfaces clean by swabbing with 5% to 10% chlorine bleach. Putfresh absorbent paper bench protectors on bench before beginning PCR.2. Wear disposable gloves and change them frequently while setting up PCRs.3. Use only sterile disposable plasticware.4. Keep a separate set of pipetting devices for setting up PCRs. If possible, use theseinstruments only with cotton-plugged tips to minimize transfer of DNA by aerosol.A separate microcentrifuge for PCR work is also desirable.5. Whenever possible, set up PCRs in a laminar-flow hood or Class II biological safetycabinet to help prevent contamination by airborne DNA particles. A UV light withinthe hood or cabinet will help inactivate contaminating DNA.6. Handle microcentrifuge tubes aseptically. Do not touch the interior of the hinged cap;if this happens, discard the tube. Microcentrifuge tubes briefly before opening topellet drops around the cap and help keep reagents and reaction mixtures away frompotentially contaminating fingers. Have only one tube open at a time, and open eachtube away from the remaining tubes. Hand-held microcentrifuge tube openers (e.g.,USA/Scientific Plastics) are available to facilitate aseptic technique.7. Include negative controls (i.e., no primer and no template) in all PCRs.
Current Protocols in Human Genetics
(2000) A.2D.1-A.2D.13Copyright © 2000 by John Wiley & Sons, Inc.Supplement 26
A.2D.1
LaboratoryGuidelines,Equipment, andStock Solutions
SPECIAL CONSIDERATIONS FOR WORKING WITH RNA
RNA is susceptible to degradation by ribonucleases, which are ubiquitous, very stable,and generally require no cofactors to function. Therefore, it is very important whenworking with RNA to take precautions against RNase contamination.1. Treat all water and salt solutions except those containing Tris with DEPC (diethyl-pyrocarbonate): add 0.2 ml DEPC per 100 ml of water or solution, shake vigorouslyto dissolve, and autoclave to inactivate remaining DEPC. DEPC inactivates ribonu-cleases by covalent modification (however, it cannot be used with Tris solutionsbecause Tris inactivates DEPC).2. If possible, make separate stock solutions to use for working with RNA and keepseparate to ensure that “dirty” pipets do not come in contact with them.3. Bake glassware 4 hr at 150
°
C. Rinse plasticware in chloroform or use directly out of the package (when it is generally free from contamination). Autoclaving will not fullyinactivate many RNases.4. Wear disposable gloves that have not been worn in any potentially RNase-contaminatedareas.
SELECTION OF BUFFERS
Table A.2D.1 reports pK
a
values for some common buffers. Note that polybasic buffers,such as phosphoric acid and citric acid, have more than one useful pK
a
value. Whenchoosing a buffer, select a buffer material with a pK
a
close to the desired working pH (atthe desired concentration and temperature for use). In general, effective buffers have arange of approximately 2 pH units centered about the pK
a
value. Ideally the dissociationconstant—and therefore the pH—should not shift with a change in concentration ortemperature. If the shift is small, as for MES and HEPES, then a concentrated stock solution can be prepared and diluted without adjustment to the pH. Buffers containingphosphate or citrate, however, show a significant shift in pH with concentration change,and Tris buffers show a large change in pH with temperature. For convenience, concen-trated stock solutions of these buffers can still be used, provided that a pH adjustment ismade
after
any temperature and concentration adjustments. All adjustments to pH shouldbe made using the appropriate base—usually NaOH or KOH, depending on the corre-sponding free counterion. Tetramethylammonium hydroxide can be used to preparebuffers without a mineral cation. Many common buffers are supplied both as a free acidor base and as the corresponding salt. By mixing precalculated amounts of each, a seriesof buffers with varying pH values can conveniently be prepared.
RECIPES
Acids, concentrated stock solutions (see Table A.2D.2) Ammonium acetate, 10 M
Dissolve 385.4 g ammonium acetate in 150 ml H
2
OAdd H
2
O to 500 mlFilter sterilize
Ammonium hydroxide, concentrated stock solution (see Table A.2D.1) ATP, 100 mM
1 g ATP (adenosine triphosphate)12 ml H
2
OAdjust pH to 7.0 with 4 M NaOHAdjust volume to 16.7 ml with H
2
OStore in aliquots indefinitely at
−
20
°
C
Supplement 26Current Protocols in Human Genetics
A.2D.2
Common Buffers,Media, and StockSolutions
Current Protocols in Human GeneticsSupplement 26
Table A.2D.1
pK
a
Values and Molecular Weights for Some Common Biological Buffers
a
NameChemical formula or IUPAC namepK
a
Useful pHrangeMW(g/mol)Phosphoric acidH
3
PO
4
2.12 (pK
a1
)—98.00Citric acid
b
C
6
H
8
O
7
(H
3
Cit)3.06 (pK
a1
)—192.1Formic acidHCOOH3.75—46.03Succinic acidC
4
H
6
O
4
4.19 (pKa
1
)—118.1Citric acid
b
C
6
H
7
O
7
−
(H
2
Cit
−
)4.74 (pKa
2
)—Acetic acidCH
3
COOH4.75—60.05Citric acid
b
C
6
H
6
O
7
−
(HCit
2
−
)5.40 (pK
a3
)—Succinic acidC
4
H
5
O
4
−
5.57 (pK
a2
)—MES2-(
N
-Morpholino]ethanesulfonic acid6.155.5-6.7195.2Bis-Trisbis(2-Hydroxyethyl)iminotris(hydroxymethyl)methane6.505.8-7.2209.2ADA
N
-(2-Acetamido)-2-iminodiaceticacid6.606.0-7.2190.2PIPESPiperazine-
N
,
N
′
-bis(2-ethanesulfonicacid)6.806.1-7.5302.4ACES
N
-(Carbamoylmethyl)-2-amino-ethanesulfonic acid6.806.1-7.5182.2Imidazole1,3-Diaza-2,4-cyclopentadiene7.00—68.08Diethylmalonic acidC
7
H
12
O
4
7.20—160.2MOPS3-(
N
-Morpholino)propanesulfonicacid7.206.5-7.9209.3Sodium phosphate,monobasicNaH
2
PO
4
7.21 (pK
a2
)—120.0Potassium phosphate,monobasicKH
2
PO
4
7.21 (pK
a2
)136.1TES
N
-tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid7.406.8-8.2229.3HEPES
N
-(2-Hydroxyethyl)piperazine-N
′
-(2-ethanesulfonic acid)7.556.8-8.2238.3HEPPSO
N
-(2-Hydroxyethyl)piperazine-
N
′
-(2-hydroxypropanesulfonic acid)7.807.1-8.5268.3Glycinamide HClC
2
H
6
N
2
O
⋅
HCl8.107.4-8.8110.6Tricine
N
-tris(Hydroxymethyl)methylglycine8.157.4-8.8179.2GlycylglycineC
4
H
8
N
2
O
3
8.207.5-8.9132.1TrisTris(hydroxymethyl)aminomethane8.307.0-9.0121.1Bicine
N
,
N
-bis(2-Hydroxyethyl)glycine8.357.6-9.0163.2Boric acidH
3
BO
3
9.24—61.83CHES2-(
N
-Cyclohexylamino)ethane-sulfonic acid9.508.6-10.0207.3CAPS3-(Cyclohexylamino)-1-propane-sulfonic acid10.409.7-11.1221.3Sodium phosphate,dibasicNa
2
HPO
4
12.32 (pK
a3
)—142.0Potassium phosphate,dibasicK
2
HPO
4
12.32 (pK
a3
)—174.2
a
Some data reproduced from
Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems
(Mohan, 1997) withpermission of Calbiochem.
b
Available as a variety of salts, e.g., ammonium, lithium, sodium.
A.2D.3
LaboratoryGuidelines,Equipment, andStock Solutions
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