STOCK SOLUTIONS, EQUIPMENT, AND APPENDIX 2

LABORATORY GUIDELINES
Common Stock Solutions, Buffers, and Media APPENDIX 2A
This collection describes the preparation of buffers and reagents used in this manual for
cell culture, manipulation of neural tissue, molecular biological methods, and neurophysi-
ological/neurochemical measurements. When preparing solutions, use deionized, dis-
tilled water and reagents of the highest available grade. Sterilization—by filtration
through a 0.22-µm filter or by autoclaving—is recommended for most applications.
Acid, concentrated stock solutions
See Table A.2A.1.
Ammonium hydroxide, concentrated stock solution
See Table A.2A.1.

Table A.2A.1 Molarities and Specific Gravities of Concentrated Acids and Basesa

Molecular Molarity 1 M solution Specific

Acid/base % by weight
weight (approx.) (ml/liter) gravity
Acetic acid (glacial) 60.05 99.6 17.4 57.5 1.05
Ammonium hydroxide 35.0 28 14.8 67.6 0.90
Formic acid 46.03 90 23.6 42.4 1.205
98 25.9 38.5 1.22
Hydrochloric acid 36.46 36 11.6 85.9 1.18
Nitric acid 63.01 70 15.7 63.7 1.42
Perchloric acid 100.46 60 9.2 108.8 1.54
72 12.2 82.1 1.70
Phosphoric acid 98.00 85 14.7 67.8 1.70
Sulfuric acid 98.07 98 18.3 54.5 1.835
aCAUTION: Handle strong acids and bases carefully.

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 liter and filter sterilize. Store up
to 6 months at room temperature.
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 increased to between 7
and 8.
Ethidium bromide staining 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 in H2O.
Ethidium bromide working solution is used to stain agarose 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 H2O for an equal Stock Solutions,
Equipment, and
continued Laboratory
Guidelines

Current Protocols in Pharmacology (1998) A.2A.1-A.2A.3 A.2A.1

Copyright © 1998 by John Wiley & Sons, Inc.
 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 mutagen and must be handled carefully.
Fetal bovine serum (FBS)
Thaw purchased fetal bovine serum (shipped on dry ice and kept frozen until
needed). Store 3 to 4 weeks at 4°C. If FBS is not to be used within this time,
aseptically divide into smaller aliquots and refreeze until used. Store ≤1 year at
−20°C. To inactivate FBS, heat serum 30 min to 1 hr in a 56°C water bath.
Repeated thawing and refreezing should be avoided as it may cause denaturation of the
serum.
Inactivated FBS (FBS that has been treated with heat to inactivate complement protein and
thus prevent an immunological reaction against cultured cells) is useful for a variety of
purposes. It can be purchased commercially or made in the lab as described above.
Gel loading buffer, 6×
0.25% (w/v) bromphenol blue
0.25% (w/v) xylene cyanol FF
40% (w/v) sucrose or 15% (w/v) Ficoll 400 or 30% (v/v) glycerol
Store at 4°C (room temperature if Ficoll is used)
This buffer does not need to be sterilized. Sucrose, Ficoll 400, and glycerol are essentially
interchangeable in this recipe.
Other concentrations (e.g., 10×) can be prepared if more convenient.
LB medium (Luria broth) and LB plates
Per liter add:
10 g tryptone
5 g yeast extract
5 g NaCl
1 ml 1 M NaOH
15 g agar or agarose (for plates only)
Autoclave solution. Add filter-sterilized additives (see below) after the solution has
cooled to 55°C. For LB plates, pour agar-containing solution into sterile petri dishes
in a tissue culture hood. Store autoclaved medium up to 1 month at room tempera-
ture. Store antibiotic-containing plates in the dark up to 2 weeks at 4°C.
Contamination is easily identified by the clouding of solutions or the presence of growth on
plates.
Additives:
Antibiotics (if required): Galactosides (if required):
Ampicillin to 50 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-
Tetracycline to 12 µg/ml galactoside (Xgal) to 20 µg/ml
Isopropyl-1-thio-β-D-galactoside (IPTG)
to 0.1 mM
Potassium phosphate buffer, 0.1 M
Solution A: 27.2 g KH2PO4 per liter (0.2 M final)
Solution B: 34.8 g K2HPO4 per liter (0.2 M final)
Referring to Table A.2A.2 for desired pH, mix the indicated volumes of solutions A
and B, then dilute with water to 200 ml. Filter sterilize if necessary. Store up to 3
months at room temperature.
Common Stock This buffer may be made as a 5- or 10-fold concentrate simply by scaling up the amount of
Solutions, Buffers potassium phosphate in the same final volume. Phosphate buffers show concentration-de-
and Media
continued
A.2A.2
Current Protocols in Pharmacology
 pendent changes in pH, so check the pH of the concentrate by diluting an aliquot to the final
concentration.
Sodium phosphate buffer, 0.1 M
Solution A: 27.6 g NaH2PO4⋅H2O per liter (0.2 M final)
Solution B: 53.65 g Na2HPO4⋅7H2O per liter (0.2 M)
Referring to Table A.2A.2 for desired pH, mix the indicated volumes of solutions
A and B, then dilute with water to 200 ml. Filter sterilize if necessary. Store up to
3 months at room temperature.
This buffer may be made as a 5- or 10-fold concentrate simply by scaling up the amount of
potassium phosphate in the same final volume. Phosphate buffers show concentration-de-
pendent changes in pH, so check the pH of the concentrate by diluting an aliquot to the final
concentration.

Table A.2A.2 Preparation of 0.1 M Sodium and Potassium Phosphate Buffersa

Desired Solution A Solution B Desired Solution A Solution B

pH (ml) (ml) pH (ml) (ml)
5.7 93.5 6.5 6.9 45.0 55.0
5.8 92.0 8.0 7.0 39.0 61.0
5.9 90.0 10.0 7.1 33.0 67.0
6.0 87.7 12.3 7.2 28.0 72.0
6.1 85.0 15.0 7.3 23.0 77.0
6.2 81.5 18.5 7.4 19.0 81.0
6.3 77.5 22.5 7.5 16.0 84.0
6.4 73.5 26.5 7.6 13.0 87.0
6.5 68.5 31.5 7.7 10.5 90.5
6.6 62.5 37.5 7.8 8.5 91.5
6.7 56.5 43.5 7.9 7.0 93.0
6.8 51.0 49.0 8.0 5.3 94.7
aAdapted by permission from CRC (1975).

TE (Tris/EDTA) buffer
10 mM Tris⋅Cl, pH 7.4, 7.5, or 8.0 (or other pH; see recipe below)
1 mM EDTA, pH 8.0 (see recipe above)
Store up to 6 months at room temperature
Tris⋅Cl, 1 M
Dissolve 121 g Tris base in 800 ml H2O
Adjust to desired pH with concentrated HCl
Adjust volume to 1 liter with H2O
Filter sterilize if necessary
Store up to 6 months at 4°C or room temperature
Approximately 70 ml HCl is needed to achieve a pH 7.4 solution, and ∼42 ml for a solution
that is pH 8.0.
IMPORTANT NOTE: The pH of Tris buffers changes significantly with temperature,
decreasing approximately 0.028 pH units per 1°C. Tris-buffered solutions should be adjusted
to the desired pH at the temperature at which they will be used. Because the pKa of Tris is
8.08, Tris should not be used as a buffer below pH ∼7.2 or above pH ∼9.0.

LITERATURE CITED Stock Solutions,

Equipment, and
Chemical Rubber Company. 1975. CRC Handbook of Biochemistry and Molecular Biology, Physical and Laboratory
Chemical Data, 3d ed., Vol. 1. CRC Press, Boca Raton, Fla. Guidelines

A.2A.3
Current Protocols in Pharmacology