APPENDIX 2
LABORATORY STOCK SOLUTIONS AND EQUIPMENT
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APPENDIX 2A
Common Buffers and Stock Solutions
This ction describes the preparation of buffers and reagents ud in the manipulation of nucleic acids.
For preparation of acid and ba stock solutions, e Tables A.2A.1 and A.2A.2 as well as individual recipes.GENERAL GUIDELINES
When preparing solutions, u 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 are volatile, altered or damaged by heat, or who pH or concentration are critical should be sterilized by filtration through a 0.22-µm filter. In many cas such components are added from concentrated stocks after the solution has been autoclaved. Where specialized sterilization methods are required, this is indicated in the individual reci
pes.
CAUTION: It is important to follow laboratory safety guidelines and heed manufacturers’precautions when working with hazardous chemicals; consult institutional safety officers and appropriate references for further details.STORAGE
Most simple stock solutions can be stored indefinitely at room temperature if reasonable care is exercid to keep them sterile; where more rigorous conditions are required, this is indicated in the individual recipes.
Current Protocols in Nucleic Acid Chemistry (2000) A.2A.1-A.2A.12Copyright © 2000 by John Wiley & Sons, Inc.
Table A.2A.1 Molarities and Specific Gravities of Concentrated Acids and Bas a
Acid/ba Molecular weight % by weight Molarity (approx.) 1 M solution (ml/liter) Specific gravity Acids
Acetic acid (glacial)60.0599.617.457.5 1.05Formic acid 46.039023.642.4 1.205 9825.938.5 1.22Hydrochloric acid 36.463611.685.9 1.18Nitric acid 63.017015.763.7 1.42Perchloric acid 100.46
60729.212.2108.882.1 1.541.70Phosphoric acid 98.008514.767.8 1.70Sulfuric acid 98.079818.354.5 1.835Bas
Ammonium hydroxide
35.02814.867.60.90Potassium hydroxide 56.114511.682.2 1.447Potassium hydroxide 56.115013.474.6 1.51Sodium hydroxide
40.0
50
19.1
52.4
1.53
a CAUTION: Handle strong acids and bas carefully.
A.2A.1
Laboratory Stock Solutions and Equipment
SPECIAL CONSIDERATIONS FOR WORKING WITH RNA
RNA is susceptible to degradation by ribonucleas, which are ubiquitous, very stable,and generally require no cofactors to function. Therefore, it is very important when working with RNA to take precautions against RNa contamination.
1. Treat all water and salt solutions except tho containing Tris with DEPC (diethyl-pyrocarbonate; e recipe below), which inactivates ribonucleas by covalent modi-fication.
2. If possible, make parate stock solutions to u for working with RNA and keep parate to ensure that “dirty” pipets do not come in contact with them.
3. Bake glassware 4 hr at 150°C. Rin plasticware in chloroform or u directly out of the package (when it is generally free from contamination). Autoclaving will not fully inactivate many RNas.
4. Wear clean disposable gloves that have not been worn in any potentially RNa-con-taminated areas.SPECIAL CONSIDERATIONS FOR PCR EXPERIMENTS
Becau the polymera chain reaction (PCR) is designed to detect very small amounts of DNA, only a few molecules of contaminating DNA will produce unwanted amplifica-tion products. Ideally, PCR should not be carried out in the same room where large quantities of DNA are handled. Even where such spatial paration is not practical, the following houkeeping procedures will help avoid contamination with extraneous DNA (H.D. Kay, pers. comm.).
设备维修保养制度1. Keep laboratory surfaces clean by swabbing with 5% to 10% chlorine bleach. Put fresh absorbent paper bench protectors on bench before beginning PCR.
2. Wear disposable gloves and change them frequently while tting up PCRs.
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3. U only sterile disposable plasticware.
4. Keep a parate t of pipetting devices for tting up PCRs. If possible, u the instruments only with cotton-plugged tips to minimize transfer of DNA by aerosol.A parate microcentrifuge for PCR work is also desirable.
5. Whenever possible, t up PCRs in a laminar-flow hood or Class II biological safety cabinet to help prevent contamination by airborne DNA particles. A UV light within the hood or cabinet will help inactivate contaminating DNA.
6. Handle microcentrifuge tubes aptically. Do not touch the interior of the hinged cap;if this happens, discard the tube. Microcentrifuge tubes briefly before opening to pellet drops around the cap and help keep reagents and reaction mixtures away from potentially contaminating fingers. Have only one tube open at a time, and open each tube away from the remaining tubes. Hand-held microcentrifuge tube openers (e.g.,USA/Scientific Plastics) are available to facilitate aptic technique.
7. Include negative controls (i.e., no primer and no template) in all PCRs.SELECTION OF BUFFERS
Table A.2A.2 reports pK a values for some common buffers. Note that polybasic buffers,such as phosphoric acid and citric acid, have more than one uful pK a value. When choosing a buffer, lect a buffer material with a pK a clo to the desired working pH (at the desired concentration and temperature for u). In general, effective buffers have a range of approximately 2 pH units centered about the pK a value. Ideally the dissociation教案范文
Current Protocols in Nucleic Acid Chemistry
A.2A.2
Common Buffers
and Stock Solutions
网关怎么设置constant—and therefore the pH—should not shift with a change in concentration or temperature. 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 containing phosphate 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 the buffers can still be ud, provided that a pH adjustment is made after any temperature and concentration adjustments. All adjustments to pH should be made using the appropriate ba—usually NaOH or KOH, depending on the corre-sponding free counterion. Tetramethylammonium hydroxide can be ud to prepare buffers without a mineral cation. Many common buffers are supplied both as a free acid or ba and as the corresponding salt. By mixing precalculated amounts of each, a ries of buffers with varying pH values can conveniently be prepared.
Table A.2A.2 pK a Values and Molecular Weights for Some Common Biological Buffers a
Name Chemical formula or IUPAC name pK a Uful pH
range
MW
(g/mol)
Phosphoric acid H3PO4 2.12 (pK a1)—98.00
Citric acid b C6H8O7 (H3Cit) 3.06 (pK a1)—192.1 Formic acid HCOOH 3.75—46.03 Succinic acid C4H6O4 4.19 (pKa1)—118.1
Citric acid b C6H7O7− (H2Cit−) 4.74 (pKa2)—
Acetic acid CH3COOH 4.75—60.05
Citric acid b C6H6O7− (HCit2−) 5.40 (pK a3)—
Succinic acid C4H5O4− 5.57 (pK a2)—
MES2-(N-Morpholino]ethanesulfonic acid 6.15 5.5-6.7195.2
Bis-Tris bis(2-Hydroxyethyl)iminotris
(hydroxymethyl)methane
6.50 5.8-
7.2209.2
ADA N-(2-Acetamido)-2-iminodiacetic
acid
6.60 6.0-
7.2190.2
PIPES Piperazine-N,N′-bis(2-ethanesulfonic
acid)
6.80 6.1-
7.5302.4
ACES N-(Carbamoylmethyl)-2-amino-
ethanesulfonic acid
6.80 6.1-
7.5182.2 Imidazole1,3-Diaza-2,4-cyclopentadiene7.00—6
8.08 Diethylmalonic acid C7H12O47.20—160.2 MOPS3-(N-Morpholino)propanesulfonic
acid
7.20 6.5-7.9209.3
Sodium phosphate,
monobasic
NaH2PO47.21 (pK a2)—120.0
Potassium phosphate,
monobasic
KH2PO47.21 (pK a2)136.1
TES N-tris(Hydroxymethyl)methyl-2-
aminoethanesulfonic acid
来之不易的意思
7.40 6.8-8.2229.3
HEPES N-(2-Hydroxyethyl)piperazine-N′-
(2-ethanesulfonic acid)
7.55 6.8-8.2238.3
HEPPSO N-(2-Hydroxyethyl)piperazine-N′-
(2-hydroxypropanesulfonic acid)
7.807.1-8.5268.3 Glycinamide HCl C2H6N2O⋅HCl8.107.4-8.8110.6 Tricine N-tris(Hydroxymethyl)methylglycine8.157.4-8.8179.2 Glycylglycine C4H8N2O38.207.5-8.9132.1
Tris Tris(hydroxymethyl)aminomethane8.307.0-9.0121.1小学生垒球
continued
Current Protocols in Nucleic Acid Chemistry
A.2A.3
RECIPES
Ammonium acetate, 10 M
Dissolve 385.4 g ammonium acetate in 150 mL H 2O Add H 2O to 500 mL Sterilize by filtration
BCIP , 5% (w/v)
Dissolve 0.5 g 5-bromo-4-chloro-3-indolyl phosphate disodium salt (stored at −20°C) in 10 mL of 100% dimethylformamide (DMF). Store wrapped in aluminum foil up to 6 months at 4°C.
The BCIP may not dissolve completely. V ortex the solution immediately before u and pipet with a wide-mouth pipet tip.Discard solution if it turns pinkish.
DEPC (diethylpyrocarbonate)-treated solutions
Add 0.2 mL DEPC to 100 mL of the solution to be treated. Shake vigorously to dissolve the DEPC. Autoclave the solution to inactivate the remaining DEPC.
CAUTION: Wear gloves and u a fume hood when using DEPC, as it is a suspected carcinogen.
Many investigators keep the solutions they u for RNA work parate to ensure that “dirty”pipets do not go into them. One may also try to have parate working area, pipettors, avoid contamination.
Do not treat solutions containing Tris with DEPC, as Tris inactivates the DEPC.
dNTPs: dATP , dTTP , dCTP , and dGTP
Concentrated stocks: Purcha deoxyribonucleoside triphosphates (dNTPs) from a commercial supplier (Pharmacia Biotech is recommended) either as ready-made 100 mM solutions, the preferred form for shipping and storage, or in lyophilized form. If purchad lyophilized, dissolve dNTPs in deionized H 2O to an expected concentration of 30 mM, then adjust to pH 7.0 with 1 M NaOH (to prevent acid-catalyzed hydrolysis). Determine the actual concentration of each dNTP by UV spectrophotometry at 260 nm, using the following extinction coefficients:adenine, εIM 260nm = 1
5,200; cytosine, εIM 260nm = 7,050; guanosine, εIM 260nm = 12,010;thymine, εIM 260nm = 8,400.
continued
Bicine N ,N -bis(2-Hydroxyethyl)glycine 8.357.6-9.0163.2Boric acid H 3BO 3
9.24—
61.83CHES 2-(N -Cyclohexylamino)ethane-sulfonic acid
9.508.6-10.0207.3CAPS
3-(Cyclohexylamino)-1-propane-sulfonic acid 10.409.7-11.1221.3Sodium phosphate,dibasic
Na 2HPO 412.32 (pK a3)—142.0Potassium phosphate,dibasic
K 2HPO 4
12.32 (pK a3)
—
174.2
a Some data reproduced from Buffers: A Guide for the Preparation and U of Buffers in Biological Systems (Mohan, 1997) with
permission of Calbiochem.
b Available as a variety of salts, e.g., ammonium, lithium, sodium.
Table A.2A.2 pK a Values and Molecular Weights for Some Common Biological Buffers a , continued
Name Chemical formula or IUPAC name pK a Uful pH range MW (g/mol)Current Protocols in Nucleic Acid Chemistry
A.2A.4
Common Buffers
and Stock Solutions
Working solutions: Prepare working solutions of desired concentration (commonly
2 mM) for each dNTP by diluting concentrated stocks appropriately. Remember that
the molarity of the 3dNTP and 4dNTP mixes refers to the concentration of each precursor prent in the solution.
4dNTP mixes: For u in various molecular biology applications, prepare mixed dNTP solutions containing equimolar amounts of all four DNA precursors; e.g.:
2 mM 4dNTP mix: 2 mM each dATP, dTTP, dCTP, dGTP
1.25 mM 4dNTP mix: 1.25 mM each of dA TP, dTTP, dCTP, dGTP.
3dNTP mixes: For u in radioactive labeling procedures, prepare similar stocks lacking one particular dNTP but containing equimolar amounts of the remaining three precursors; e.g.:
2 mM 3dNTP mix (minus dA TP): 2 mM each of dTTP, dCTP, dGTP.
Store dNTPs and dNTP mixtures as aliquots at −20°C (stable for ≤1 year).
DTT (dithiothreitol), 1 M
Dissolve 1.55 g DTT in 10 mL water and filter sterilize. Store in aliquots at −20°C.
Do not autoclave to sterilize.
EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0)
Dissolve 186.1 g disodium EDTA dihydrate in 700 mL water. Adjust pH to 8.0 with
10 M NaOH (∼50 mL; add slowly). Add water to 1 L and filter sterilize.
Begin titrating before the sample is completely dissolved. EDTA, even in the disodium salt form, is difficult to dissolve at this concentration unless the pH is incread to between 7 and 8. Heating the solution may also help to dissolve EDTA.
Ethidium bromide solution
Concentrated stock (10 mg/mL): Dissolve 0.2 g ethidium bromide in 20 mL H2O.
Mix well and store at 4°C in dark or in a foil-wrapped bottle. Do not sterilize.
Working solution: Dilute stock to 0.5 µg/mL or other desired concentration in electrophoresis buffer (e.g., 1× TBE or TAE) or water.
To u: Ethidium bromide working solution is ud to stain agaro gels to permit visualization of nucleic acids under UV light. Gels should be placed in a glass dish containing sufficient working solution to cover them and shaken gently or allowed to stand for 10 to 30 min. If necessary, gels can be destained by shaking in electrophoresis buffer or water for an equal length of time to reduce background fluorescence and facilitate visualization of small quantities of DNA.
Alternatively, a gel can be run directly in ethidium bromide by using working solution (made with electrophoresis buffer) as the solvent and running buffer for the gel.
CAUTION: Ethidium bromide is a toxic and powerful mutagen. Gloves should be worn when working with solution or gel and a mask should be worn when weighing out solid. Keep parate solid and liquid waste containers for disposal of ethidium bromide–contaminated material.
Formamide loading buffer, 2×
Prepare in deionized formamide:
0.05% (w/v) bromphenol blue
0.05% (w/v) xylene cyanol FF
20 mM EDTA
Do not sterilize
Store at −20°C
Current Protocols in Nucleic Acid Chemistry A.2A.5 Laboratory Stock Solutions and Equipment
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