Buffers and Standard Solutions http://ivaan.com/protocols/151.

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BUFFERS AND STANDARD SOLUTIONS

Last Update: December 2006
from Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory)

BUFFER AND SOLUTION PREPARATION

Mix 29 g of acrylamide, 1 g of N, N'-methylenebisacrylamide, and 60 mL of dH2 O. check pH (<7.0). Filter sterilize
30% Acrylamide (60 mL):
(0.45m) Store at 4oC in the dark
0.1M ATP (1 mL): Mix 60 mg of ATP with 0.8 mL of dH2 O. pH to 7.0 with 0.1M NaOH. Add dH2O to 1 mL. Store at -70o C
10M Ammonium acetate (1 L): Mix 770 g of ammonium acetate and 800 mL dH2 O. add dH2O to 1 L and filter sterilize (0.45m)
10% Ammonium persulfate (APS): Mix 1 g ammonium persulfate and dH2 O to 10 mL. Store at 4oC
Mix 0.5 g of 5-bromo-4-chloro-3-indolyl phosphate disodium salt with 10 mL of 100% dimethylformamide. Store at
BCIP (10 mL):
4o C in the dark
1M CaCl2 (200 mL): Mix 54 g of CaCl2 -6H2O with 200 mL of dH2 O. Filter sterilize (0.22m) Store at -20oC
2.5M CaCl2 (20 mL): Mix 13.5 g CaCl2-6H2 O with 20 mL dH2 O. Filter sterilize (0.22m) Store at -20o C
Dissolve in dH2 O to 100mM. pH to 7.0 with 0.05M Tris base. Calculate concentration using OD. Dilute to 50mM
o 4 -1
Deoxyribonucleotide triphosphates with dH2 O. Store at -70 C. Use a cuvette with a path length of 1 cm: A: wavelength = 259 nm, e = 1.54 x 10 M
(dNTPs): cm-1 . G: wavelength = 253 nm, e = 1.37 x 104 M-1 cm-1 . C: wavelength = 271 nm, e = 9.10 x 103 M-1 cm-1 . T:
wavelength = 260 nm, e = 7.40 x 103 M-1 cm-1 . Absorbance = eM
1M Dithiothreitol (DTT) (20 mL): Mix 3.09 g of DTT with 20 mL of 0.01M sodium acetate pH 5.2. Filter sterilize (0.22m) Store at -20o C
Mix 186.1 g of disodium ethylenediaminetetraacetate-2H2 O with 800 mL of dH2 O. pH 8.0 with ~20 g NaOH
0.5M EDTA pH 8.0:
(Autoclave)
10 mg/mL Ethidium bromide: Mix 1 g of ethidium bromide with 100 mL of dH2 O. Stirr ON. Store in the dark at RT
Mix 1.6 g of NaCl, 0.074 g of KCl, 0.027 g of Na2HPO4 -2H2 O, 0.2 g of dextrose, 1 g HEPES, and 90 mL of dH2 O.
2X Hepes-buffered saline:
Filter sterilize (0.22m) Store at -20o C
1M Isopropylthio-b-D-galactoside
Mix 2 g of IPTG, and 8 mL of dH2 O. Adjust 10 mL w/ dH2 O. Filter sterilize (0.22m) Store at -20o C
(IPTG) (10 mL):
1M Magensium acetate (1 L): Mix 214.46 g of magnesium acetate-4H2O with 800 mL of dH2 O. Add dH2O to 1 L. Filter sterilize (0.45m)

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1M MgCl2 (1 L): Mix 203.3 g of MgCl3 -6H2 O with 800 mL dH2 O. Add dH2 O to 1 L (Autoclave)
b-Mercaptoethanol (BME): Ususally obtained as a 14.4M solution. Store in dark at 4o C
Nitro blue tetrazolium chloride
(NBT) (10 mL): Mix 0.5 g of NBT with 10 mL of 70% dimethylformamide. Store at 4o C in the dark

Phenol:chloroform: Mix 1:1 phenol:chloroform. Extract X times with 0.1M Tris HCl pH 7.6. Store under 0.01M Tris HCl pH 7.6 at 4o C
in the dark
10mM Phenylmethylsulfonyl Mix 1.74 mg/mL of PMSF in isopropanol. Store at -20o C (long term). T1/2 of 35 mins at pH 8.0 for a 20uM
fluoride (PMSF): solution. Inactivation faster at 25o C than at 4o C
Phosphate-buffered saline (PBS) (1 Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2 HPO4, 0.24 g of KH2PO4, and 800 mL of dH2 O. Add dH2 O to 1 L
L): (Autoclave). Store at RT
1M Potassium acetate, pH 7.5 (100 Mix 9.82 g of potassium acetate with 90 mL dH2 O. pH to 7.5 w/ 2M acetic acid. Add dH2 O to 100 mL. Store at
mL): -20o C
Potassium acetate (for alkaline lysis)
Mix 60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid, and 28.5 mL dH2 O
(3M potassium/5M acetate):
Mix 408.1 g of sodium acetate-3H2 O with 800 mL of dH2O. pH to 5.2/7.0 with glacial acetic acid. Add dH2 O to 1 L
3M Sodium acetate (pH 5.2/7.0):
(Autoclave)
5M NaCl (1 L): Mix 292.2 g of NaCl with 800 mL of dH2 O. Add dH2 O to 1 L (Autoclave)
10 % Sodium dodecyl sulfate (SDS)
(also called sodium lauryl sulfate) Mix 100 g of SDS with 900 mL of dH2 O. Heat to 68oC. pH to 7.2 w/ cHCl. Add dH2 O to 1 L
(1 L):
5 M NaOH (1 L): Mix 200 g of NaOH and dH2 O to 1 L
Mix 175.3 g of NaCl, 88.2 g of sodium citrate, and 800 mL dH2O. pH to 7.0 with 10N NaOH. Add dH2 O to 1 L
20X SSC (1 L):
(Autoclave)
Mix 175.3 g of NaCl, 27.6 g of NaH2 PO4 -H2 O, 7.4 g of EDTA, and 800 mL dH2O. pH to 7.4 w/ NaOH (~6.5 mL of
20X SSPE (1 L):
a 10N solution). Add dH2 O to 1 L (Autoclave)
Trichloroacetic acid (TCA) 100%
Mix 500 g of TCA with 227 mL dH2O
solution (500 mL):
Mix 121.1 g of Tris base with 800 mL dH2 O. Add 70 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1o C
1M Tris, pH 7.4 (1 L):
increase. Make sure it is at RT before making final pH adjustments. Add dH2 O to 1 L (Autoclave)
Mix 121.1 g of Tris base with 800 mL dH2 O. Add 60 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1o C
1M Tris, pH 7.6 (1 L):
increase. Make sure it is at RT before making final pH adjustments. Add dH2 O to 1 L (Autoclave)

1M Tris, pH 8.0 (1 L): Mix 121.1 g of Tris base with 800 mL dH2 O. Add 42 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1o C
increase. Make sure it is at RT before making final pH adjustments. Add dH2 O to 1 L (Autoclave)
Tris-buffered saline (TBS) (25 mM Mix 8 g of NaCl, 0.2 g of KCl, 3 g of Tris base, and 800 mL of dH2 O. Add 0.015 g Phenol red and pH to 7.4 with
Tris) (1 L): HCl. Add dH2 O to 1 L (Autoclave)

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Buffers and Standard Solutions
X-gal (1 mL):
50X Denhardts (500 mL):
10 mg/mL Salmon Sperm DNA:
RNAse (DNAse free):
TE, pH 7.4 (100 mL):
TE, pH 7.6 (100 mL):
TE, pH 8.0 (100 mL):
STE (TEN) (100 mL):
STET (100 mL):
TNT (100 mL):
50X TAE (Tris acetate) (1 L):
10X TPE (Tris phosphate) (1 L):
5X TBE (Tris borate) (1 L):
1X Alkaline (freshly made) (1 L):
5X Tris glycine (50 mL):
SOC medium (100 mL):
1M Potassium Phosphate (100 mL):
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Mix 20 mg of 5-bromo-4-chloro-3-indolyl-b-D-galactoside with 1 mL of dimethylformamide (use polypropylene
tube). Store in the dark at -20o C
Mix 5 g of Ficoll-400, 5 g of Polyvinylpyrrolidone, 5 g of Bovine serum albumin Type V, and dH2 O to 500 mL
Mix 100 mg of Salmon Sperm DNA (Type III) with 10 mL dH2 O until it dissolves. Add 0.058 g of NaCl (0.1M).
Phenol extract, followed by a phenol:chloroform extraction. Shear DNA by passing it (>12 times) through a 17-gauge
neddle. Add 20 mL of ice cold ethanol to precipitate DNA. Measure OD260 . Boil for 10 mins and store at -20o C. Boil
in a water bath for 5 minutes and chill on ice before use
Mix 10 mg of RNAse A (pancreatic) with 10 mL 10mM Tris pH 7.5, 15mM NaCl. Heat to 100oC for 15 minutes.
Allow to cool to RT (ON). Store at -20o C
Mix 1 mL of 1M Tris-HCl, pH 7.4 (10mM), 200 mL of 0.5M EDTA, pH 8.0 (1mM), and 98.8 mL of dH2 O
Mix 1 mL of 1M Tris-HCl, pH 7.6 (10mM), 200 mL of 0.5M EDTA, pH 8.0 (1mM), and 98.8 mL of dH2 O
Mix 1 mL of 1M Tris-HCl, pH 8.0 (10mM), 200 mL of 0.5M EDTA, pH 8.0 (1mM), and 98.8 mL of dH2 O
Mix 2 mL of 5M NaCl (100mM), 1 mL of 1M Tris-HCl, pH 8.0 (10 mM), 200 mL of 0.5M EDTA, pH 8.0 (1mM)
and 96.8 mL of dH2 O
Mix 2 mL of 5M NaCl (100mM), 1 mL of 1M Tris-HCl, pH 8.0 (10 mM), 200 mL of 0.5M EDTA, pH 8.0 (1mM),
10 mL of 50% Triton X-100 (5%), and 86.8 mL of dH2 O
Mix 1 mL of Tris-HCl, pH 8.0 (10mM), 3 mL of of 5M NaCl (150mM), 5 mL of 100% Tween 20 (0.05%), and 91
mL of dH2O
Mix 242 g of Tris base, 57.1 mL of glacial acetic acid, 100 mL of 0.5M EDTA, pH 8.0, and dH2O to 1 L
Mix 108 g of Tris base, 15.5 mL of 85% phosphoric acid (1.679 g/mL), 10 mL of 0.5M EDTA, pH 8.0, and dH2 O to
1L
Mix 54 g of Tris base, 27.5 g of boric acid, 20 mL of 0.5M EDTA, pH 8.0, and dH2 O to 1 L
Mix 5 mL of 10N NaOH, 2 mL of 0.5M EDTA, pH 8.0, and dH2 O to 1 L
Mix 15.1 g of Tris base, 94 g of glycine, pH 8.3, and 50 mL 10% SDS
Mix 2 g of bacto-tryptone, 0.55 g of yeast extract, 1 mL of 1M NaCl, 1 mL of 1M KCl, and dH2 O to 100 mL
(Autoclave). Let cool to 55o C. Add 1 mL of 1M MgCl2, 1 mL of 1M MgSO4, and 1 mL 2M glucose. Filter through a
0.22m filter
pH Volume of 1M K2 HPO4 (mL) Volume of KH2 PO4 (mL)
5.8 8.5 91.5
6.0 13.2 86.8
6.2 19.2 80.8
6.4 27.8 72.2
6.6 38.1 61.9
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6.8 49.1 50.3

7.0 61.5 38.3
7.2 71.7 28.3
7.4 80.2 19.8
7.6 86.6 13.4
7.8 90.8 9.2
8.0 94.0 6.0
pH Volume of Na2 HPO4 (mL) Volume of NaH2 PO4 (mL)
5.8 7.9 92.1
6.0 12.0 88.0
6.2 17.8 82.2
6.4 25.5 74.5
6.6 35.2 64.8
1M Sodium Phosphate (100 mL): 6.8 46.3 53.7
7.0 57.7 42.3
8.2 68.4 31.6
7.4 77.4 22.6
7.6 84.5 15.5
7.8 89.6 10.4
8.0 93.2 6.8

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