Molecular Biology Buffers And Solutions

NOTE: Use ACS grade chemicals, and dH2O for all buffers.
Use Sigma Trizma Tris Base and Tris-HCl for all buffers.
10 X TE: 500 ml
6.1 g
Tris Base
1.9 g
Na2EDTA-2H2O
Adjust to pH 8.0 with HCl.
Autoclave.

1X Conc.
10 mM
1 mM

3M KOAc, pH 5.0: 500 ml

148 g KOAc
Add 100 ml dH2O and mix
Add 100 ml AcCOOH
Measure pH and add AcCOOH to pH 5.0 (About 200-250 ml of AcCOOH).
Add dH2O to about 450 ml.
Cool to room temperature and adjust pH to 5.0.
Bring to final volume with dH2O.
3M NaOAc, pH 5.0: 500 ml
204.1 g NaOAc-3H2O
Add 100 ml dH2O and mix
Add 100 ml AcCOOH
Measure pH and add ACOOH to pH 5.0 (About 1-10 ml of AcCOOH).
Add dH2O to about 450 ml.
Cool to room temperature and adjust pH to 5.0.
Bring to final volume with dH2O.
10 M NH4OAc, pH ~7: 50 ml
38.5 g NH4OAc
Add dH2O to 50 ml in glass cylinder.
Warm to ~50C (microwave).
Do not measure or adjust pH.
Filter sterilize (0.45 m).
20X SSC or SSCP: 1 L
20X Conc.
175.3 g NaCl
3M
88.2 g
Na3Citrate-2H2O
0.3 M
31.2 g
NaH2PO4-2H2O
0.2 M
Add dH2O to 900 ml
SSC: Add 1M HCl to pH 6.6. SSCP: Add NaOH to pH 6.6.
Dilute 10X and measure pH. pH should be ~7.2.
Add dH2O to 1 L.
NaCl + PEG Solution: 100 ml
Final
9.35 g
NaCl
1.6 M
13 g
Polyethylene glycol 8000
13% (w/v)
Bring to 100 ml with dH2O. Filter sterilize.

1X Conc.
150 mM
15 mM
10 mM

50 mM GTE: 500 ml
1.5 g
Tris-Base
1.9 g
Na2EDTA-2H2O
4.5 g
Dextrose
Add HCl to pH 8.0.
Autoclave.

1X Conc.
25 mM Tris
10 mM EDTA
50 mM glucose

50X Denhardts Solution

1% Ficoll-400
1% Polyvinylpyrrolidone (PVP)
1% Bovine serum albumin (BSA)
SM: 1 L
5.8 g
2.0 g
6.4 g
1.2 g
0.2 g
Autoclave

1X Conc.
NaCl
MgSO4-7H2O
Tris-HCl
Tris-Base
Gelatin

50 mM Tris, pH 7.5

10X Ligase Buffer: 1 ml

700 l
0.5 M Tris-HCl, pH 7.5
100 l
1M MgCl2
100 l
1M DTT
50 l
100 mM ATP (kept at -80C)
50 l
NEB Acetylated BSA (10 mg/ml)

(We normally aliquot the 10X ligase

buffer from NEB, and store at -70C.)

Heat Treated RNase A Aliquots

Use RNase Type IIA from Sigma (R-5000).
1. Dissolve 100 mg RNase A in 5 ml of: 10 mM Tris, pH 7.5, 1 mM EDTA, 15 mM NaCl
2. Place in boiling water bath for 15 minutes.
3. Cool slowly to room temperature.
4. Add 5 ml of glycerol and mix completely.
5. Store 1 ml aliquots at -20C.
6. Use sterile pipet tips when removing solution.
Proteinase K Stock Solution Aliquots
Use Proteinase K from BMB, GIBCO/BRL, or Sigma.
1. Dissolve 50 mg proteinase K in 0.5 ml 10 mM Hepes-NaOH, pH 7.5, 1 mM CaCl2.
2. Self-digest 15 minutes at 37C
3. Add 0.5 ml 50% glycerol.
2. Divide into 10 X 100 l aliquots. Store at -20C.
3. Use sterile pipet tips when removing solution.
Proteinase K tolerates: 1% SDS, 4 M urea, up to 65C.
Carbenicillin or Ampicillin Stock Aliquots
1. Dissolve 1.0 g of carbenicillin or ampicillin (sodium salt) in 10 ml of dH2O.
2. Filter sterilize with syringe into 10 sterile microfuge tubes. Label AMP, 1000X.

CaCl2 solution for competent cells: 200 ml

30 g
Glycerol
0.76 g
Pipes (dipotassium) (378.5 g/mol; 10 mM)
1.76 g
CaCl2-2H2O (147.0 g/mol; 60 mM)
Add HCl to pH 7.0. Divide into 100 ml aliquots. Autoclave.
Vanadyl Adenosine Complex (VAC): 30 ml
1. Prepare 2 M vanadyl sulfate (Aldrich 20,486-2): 1.74 g in 4 ml dH2O.
2. Prepare 250 mM adenosoine (Sigma A-9251): 2 g in 30 ml dH2O. Heat to near boiling while
stirring to dissolve. Once in solution, adjust to 30 ml.
3. To 24 ml adenosine, slowly add 3 ml vanadyl sulfate solution while continuing to heat and stir to
prevent precipitation.
4. Adjust pH to 6.0 with 10 N NaOH. Then pH to 7.0 with 1 N NaOH. Solution will clear at pH 6.0
and remain clear. Use pH paper for hot solution.
5. Adjust final volume to 30 ml. Aliquot in 1 ml portions. Keep hot and stirred. Store at -80C.
Final concentration in 200 mM.
Protease Inhibitor Cocktails (PICs)
1000X "D" in DMSO:
0.88 g
PMSF (0.5M)
Sigma P 7626
10 mg
Pepstatin A
BMB 1359 053 (10 mg; use all)
10 mg
Chymostatin
BMB 1004 638 (10 mg; use all)
Add DMSO to 10 ml. Dissolve. Store at -20C in 1 ml aliquots.
1000X "W" in water:
1.57 g
Benzamidine (1M)
Sigma B 6506
5 mg
Leupeptin
Sigma L 2884 (5 mg; use all)
5 mg
Bestatin
Sigma B 8385 (5 mg; use all)
Add dH2O to 10 ml. Dissolve. Store at -20C in 1 ml aliquots.
Mountant Medium for Immunofluorescence: 10 ml
10 mg
p-phenylene diamine (pPDA)
1 ml
100 mM TrisHCl, pH 8.5 (at 25C)
11.35 g Glycerol (9 ml)
Dissolve pPDA in Tris buffer (bath sonication helps). Shake with glycerol in 50 ml tube to mix.
Centrifuge to remove bubbles. Aliquot 9 X 1 ml in 1.5 ml tubes and 10 X 100 l in 0.5 ml tubes.
Store at -70C. Note: pH must be >8.0. See Biotechniques, 20:398-400, March 1996.
Protein Assay Reagent A1: 500 ml
50 g
Na2CO3
10 g
NaOH
0.5 g
Potassium Tartrate
Add dH2O to 500 ml
Store in tightly capped plastic bottle.

Protein Assay Reagent A2: 50 ml

0.25 g CuSO4
Add dH2O to 50 ml
Store in tightly capped plastic bottle.