BUFFERS AND STANDARD SOLUTIONS |
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Last Update: December 2006 |
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from Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory) |
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| BUFFER AND SOLUTION PREPARATION | ||||||||||||||||||||||||||||||||||||||||
| 30% Acrylamide (60 mL): | Mix 29 g of acrylamide, 1 g of N, N'-methylenebisacrylamide, and 60 mL of dH2O. check pH (<7.0). Filter sterilize (0.45m)
Store at 4oC in the dark
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| 0.1M ATP (1 mL): | Mix 60 mg of ATP
with 0.8 mL of dH2O. pH to 7.0 with 0.1M NaOH. Add dH2O to 1 mL. Store at -70oC
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| 10M Ammonium acetate (1 L): | Mix 770 g of ammonium acetate and 800 mL dH2O. add dH2O to 1 L
and filter sterilize (0.45m) |
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| 10% Ammonium persulfate (APS): | Mix 1 g ammonium persulfate
and dH2O to 10 mL. Store at 4oC |
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| BCIP (10 mL): | Mix 0.5 g of 5-bromo-4-chloro-3-indolyl phosphate disodium salt
with 10 mL of 100% dimethylformamide. Store at 4oC in the dark
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| 1M CaCl2 (200 mL): | Mix 54 g of CaCl2-6H2O
with 200 mL of dH2O. Filter sterilize (0.22m) Store at -20oC |
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| 2.5M CaCl2 (20 mL): | Mix 13.5 g CaCl2-6H2O
with 20 mL dH2O. Filter sterilize (0.22m) Store at -20oC |
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| Deoxyribonucleotide triphosphates (dNTPs): | Dissolve in dH2O to 100mM. pH to 7.0 with 0.05M Tris base. Calculate concentration using OD. Dilute to 50mM with dH2O. Store at -70oC. Use a cuvette with a path length of 1 cm: A: wavelength = 259 nm, e = 1.54 x 104 M-1 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 |
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| 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 -20oC |
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| 0.5M EDTA pH 8.0: | Mix 186.1 g of disodium ethylenediaminetetraacetate-2H2O with 800 mL of dH2O. pH 8.0 with ~20 g NaOH
(Autoclave)
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| 10 mg/mL Ethidium bromide: | Mix 1 g of ethidium bromide
with 100 mL of dH2O. Stirr ON. Store in the dark at RT
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| 2X Hepes-buffered saline: | Mix 1.6 g of NaCl, 0.074 g of KCl, 0.027 g of Na2HPO4-2H2O, 0.2 g of dextrose, 1 g HEPES, and 90 mL of dH2O. Filter sterilize (0.22m) Store at -20oC |
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| 1M Isopropylthio-b-D-galactoside (IPTG) (10 mL): | Mix 2 g of IPTG, and 8 mL of dH2O. Adjust 10 mL w/ dH2O. Filter sterilize (0.22m) Store at -20oC |
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| 1M Magensium acetate (1 L): | Mix 214.46 g of magnesium acetate-4H2O with 800 mL of dH2O. Add dH2O to 1 L. Filter sterilize (0.45m) |
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| 1M MgCl2 (1 L): | Mix 203.3 g of MgCl3-6H2O
with 800 mL dH2O. Add dH2O to 1 L (Autoclave) |
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| b-Mercaptoethanol (BME): | Ususally obtained as a 14.4M solution. Store in dark at 4oC
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| Nitro blue tetrazolium chloride (NBT) (10 mL): | Mix 0.5 g of NBT with 10 mL of 70% dimethylformamide. Store at 4oC in the dark
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| 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 4oC in the dark
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| 10mM Phenylmethylsulfonyl fluoride (PMSF): | Mix 1.74 mg/mL of PMSF in isopropanol. Store at -20oC (long term). T1/2 of 35 mins at pH 8.0 for a 20uM solution. Inactivation faster at 25oC than at 4oC
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| Phosphate-buffered saline (PBS) (1 L): | Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, and 800 mL of dH2O. Add dH2O to 1 L (Autoclave). Store at RT |
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| 1M Potassium acetate, pH 7.5 (100 mL): | Mix 9.82 g of potassium acetate
with 90 mL dH2O. pH to 7.5 w/ 2M acetic acid. Add dH2O to 100 mL. Store at -20oC
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| Potassium acetate (for alkaline lysis) (3M potassium/5M acetate): | Mix 60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid, and 28.5 mL dH2O |
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| 3M Sodium acetate (pH 5.2/7.0): | Mix 408.1 g of sodium acetate-3H2O with 800 mL of dH2O. pH to 5.2/7.0 with glacial acetic acid. Add dH2O to 1 L (Autoclave) |
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| 5M NaCl (1 L): | Mix 292.2 g of NaCl
with 800 mL of dH2O. Add dH2O to 1 L (Autoclave) |
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| 10 % Sodium dodecyl sulfate (SDS) (also called sodium lauryl sulfate) (1 L): | Mix 100 g of SDS
with 900 mL of dH2O. Heat to 68oC. pH to 7.2 w/ cHCl. Add dH2O to 1 L |
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| 5 M NaOH (1 L): | Mix 200 g of NaOH and dH2O to 1 L |
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| 20X SSC (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 dH2O to 1 L (Autoclave) |
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| 20X SSPE (1 L): | Mix 175.3 g of NaCl, 27.6 g of NaH2PO4-H2O, 7.4 g of EDTA, and 800 mL dH2O. pH to 7.4 w/ NaOH (~6.5 mL of a 10N solution). Add dH2O to 1 L (Autoclave) |
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| Trichloroacetic acid (TCA) 100% solution (500 mL): | Mix 500 g of TCA with 227 mL dH2O |
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| 1M Tris, pH 7.4 (1 L): | Mix 121.1 g of Tris base
with 800 mL dH2O. Add 70 mL cHCl. pH is temperature dependent: ~0.03 pH units per
1oC increase. Make sure it is at RT before making final
pH adjustments. Add dH2O to 1 L
(Autoclave) |
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| 1M Tris, pH 7.6 (1 L): | Mix 121.1 g of Tris base with 800 mL dH2O. Add 60 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1oC increase. Make sure it is at RT before making final pH adjustments. Add dH2O to 1 L (Autoclave) |
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| 1M Tris, pH 8.0 (1 L): | Mix 121.1 g of Tris base with 800 mL dH2O. Add 42 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1oC increase. Make sure it is at RT before making final pH adjustments. Add dH2O to 1 L (Autoclave) |
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| Tris-buffered saline (TBS) (25 mM Tris) (1 L): | Mix 8 g of NaCl, 0.2 g of KCl, 3 g of Tris base, and 800 mL of dH2O. Add 0.015 g Phenol red and pH to 7.4 with HCl. Add dH2O to 1 L (Autoclave) |
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| X-gal (1 mL): | 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 -20oC
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| 50X Denhardts (500 mL): | Mix 5 g of Ficoll-400, 5 g of Polyvinylpyrrolidone, 5 g of Bovine serum albumin Type V, and dH2O to 500 mL
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| 10 mg/mL Salmon Sperm DNA: | Mix 100 mg of Salmon Sperm DNA (Type III)
with 10 mL dH2O 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 -20oC. Boil in a water bath for 5 minutes and chill on ice
before use |
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| RNAse (DNAse free): | 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 -20oC
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| TE, pH 7.4 (100 mL): | 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 dH2O |
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| TE, pH 7.6 (100 mL): | 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 dH2O |
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| TE, pH 8.0 (100 mL): | 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 dH2O |
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| STE (TEN) (100 mL): | 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 dH2O |
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| STET (100 mL): | 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 dH2O |
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| TNT (100 mL): | 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 |
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| 50X TAE (Tris acetate) (1 L): | 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
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| 10X TPE (Tris phosphate) (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 dH2O to 1 L
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| 5X TBE (Tris borate) (1 L): | Mix 54 g of Tris base, 27.5 g of boric acid, 20 mL of 0.5M EDTA, pH 8.0, and dH2O to 1 L
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| 1X Alkaline (freshly made) (1 L): | Mix 5 mL of 10N NaOH, 2 mL of 0.5M EDTA, pH 8.0, and dH2O to 1 L
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| 5X Tris glycine (50 mL): | Mix 15.1 g of Tris base, 94 g of glycine, pH 8.3, and 50 mL 10% SDS
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| SOC medium (100 mL): | Mix 2 g of bacto-tryptone, 0.55 g of yeast extract, 1 mL of 1M NaCl, 1 mL of 1M KCl, and dH2O to 100 mL (Autoclave). Let cool to 55oC. Add 1 mL of 1M MgCl2, 1 mL of 1M MgSO4, and 1 mL 2M glucose. Filter through a 0.22m filter |
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| 1M Potassium Phosphate (100 mL): |
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| 1M Sodium Phosphate (100 mL): |
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