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常用缓冲液的配置，欢迎补充
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PHEM (500 mls) 2x 18.14 g Pipes 6.5 g Hepes 3.8 g EGTA 0.99 g MgSO4 pH 7.0 w/ KOH PBS (5x in 500 mls) 20.45 g NaCl 0.465 g KCl 10.142 g Na2 HPO4 *7 H2 O 0.545 g KH2 PO4 pH 7.2 Mounting Media 20mM Tris pH 8.0 0.5% N-propyl gallate 90% Glycerol Store at 4oC PMBuffer* 100 ml 1 M PIPES (100 mM PIPES pH= 6.9) 2 ml 1 M MgSO4 , (2 mM MgSO4 ) 2 ml 0.5 M EGTA (1 mM EGTA,) distilled water to 900 ml adjust pH to 6.9 distilled water to 1 liter store at 4oC Valap* Put 50 g Vaseline, 50 g lanolin, and 50 g paraffin (all available from Fisher) in a 1 L Pyrex beaker Heat on "low" setting on a hotplate, stirring occasionally, until all components are melted and well mixed Pour into several small screw-cap jars (~50 ml capacity) Store at room temperature 20 x Energy Regeneration System* 150 mM creatine phosphate (Boehringer #127574) 2 ml of 100 mM MgATP Stock (20 mM ATP, 20 mM MgSO4 ) 40ml 0.5 M EGTA (2 mM EGTA) distilled water to 10 ml Disburse into 100 ml aliquots and store at -20oC Isolation Buffer* 20 ml 5 M NaCl Stock (0.1 M NaCl) 4 ml of 1 M MgSO4 Stock (4 mM MgSO4 ) 2 ml of 0.5 M EDTA (1mM EDTA) 10 ml of 1 M HEPES Stock (10 mM HEPES) distilled water to 900 ml adjust pH to 7.0 distilled water to 1 liter store at 4oC High Salt Buffer* 120 ml of 5 M NaCl Stock (0.6 M NaCl) 4 ml of 1 M MgSO4 Stock(4 mM MgSO4 ) 2 ml of 0.5 M EDTA (1mM EDTA) 10 ml of 1 M HEPES Stock (10 mM HEPES) distilled water to 900 ml adjust pH to 7.0 distilled water to 1 liter store at 4oC 10 x Column Buffer* 500 ml 1 M PIPES Stock (250 mM PIPES) 40 ml of 0.5 M EGTA Stock (10 mM EGTA) 40 ml of 1 M MgSO4 Stock (5 mM MgSO4 ) distilled water to 1800 ml adjust pH to 6.7 distilled water to 2 liters Store at 4oC Homogenization Buffer* 300 ml PM buffer 52.3 mg PMSF (1mM PMSF) 3 g leupeptin (10 mg/ml leupeptin) 300 mg pepstatin A (1 ug/ml pepstatin A) 3 mg TAME (10 ug/ml TAME) mix well to dissolve, use immediately PMG Buffer* 80 ml 1 M PIPES Stock (80 mM PIPES) 2 ml 1 M MgSO4 Stock, (2 mM MgSO4 ) 2 ml 0.5 M EGTA Stock (1 mM EGTA,) 600 ml Glycerol distilled water to 900 ml, mix well adjust pH to 6.9 distilled water to 1 liter store at 4oC PBS* 8.18 g NaCl(140 mM NaCl) 0.186 g KCl (2.5 mM KCl) 0.218 g KH2 PO4 (1.6 mM KH2 PO4 ) 2.15 g Na2 HPO4 (15 mM Na2 HPO4 ) distilled water to 1 liter Store at room temperature 1M MgSO4 Stock* 24.074 g MgSO4 distilled water to 200 ml store at room temperature 1 M HEPES, pH = 7.0Stock* 119.15 g HEPES (free acid) distilled water to 400 ml add solid NaOH a few pellets at a time while mixing until the pH is ~6.8 add concentrated NaOH dropwise to achieve pH = 7.0 distilled water to 500 ml sterile filter and store at 4oC 1 M PIPES, pH = 6.9Stock* 151.2 g PIPES (free acid) distilled water to 400 ml add solid NaOH a few pellets at a time while mixing until the pH is ~6.7 add concentrated NaOH dropwise to achieve pH = 6.9 distilled water to 500 ml sterile filter and store at 4oC 0.5 M EDTA Stock* 16.81 g EDTA (Sodium Salt) distilled water to 90 ml Adjust pH to 7.0 Distilled water 100 ml Store at room temperature 0.5 M EGTA Stock* 19.02 g EGTA (Sodium Salt) distilled water to 90 ml Adjust pH to 7.0 Distilled water to 100 ml Store at room temperature 1M dithiothreitol (DTT)* 1.542 g dithiothreitol distilled water to 10 ml disburse into 500 ml aliquots and store at -20oC 1 M KCl Stock* 74.55 g KCl distilled water to 1 liter store at room temperature 10 M NaOH Stock* 40 g NaOH distilled water to 100 ml store at room temperature 100 mM MgGTP Stock* check formula weight of the lot of GTP you have, determine the amount required for 10 mlof a 100 mM solution. add 8.5 ml distilled water to the determined amount add 1 ml of 1M MgSO4 Stock adjust pH to 7.0, distilled water to 10 ml disburse into 200 ml aliquots, store at -20oC 100 mM MgATP Stock* check formula weight of the lot of ATP you have, determine the amount required for 10 ml of a 100 mM solution. add 8.5 ml distilled water to the determined amount add 1 ml of 1M MgSO4 Stock adjust pH to 7.0 distilled water to 10 ml disburse into 200 ml aliquots, store at -20oC 5 M NaCl Stock* 292.2 g NaCl distilled water to 1 liter store at room temperature 3.6 mM Brefeldin A* 10 mg Brefeldin A (available from Epicenter Technologies Madison, WI) absolute ethanol to 12 ml store at -20oC 30 x NaF* 378 mg NaF (0.9 M NaF) distilled water to 10 ml Store at -20oC 30 x AlCl3 * 20 mg (1.5 mM AlCl3 ) distilled water to 100 ml Store at room temperature 30 mM Mg GTP-g-S* 10 mg GTP-g-S tetralithium salt (available from Boehringer Mannheim #220 467) 18 ml 1 M MgSO4 Stock (30 mM MgSO4 ) add 572 ml distilled water (to 592 ml) Store at -20oC 150 mM Mg AMP-PNP* 25 mg AMP-PNP (available from Boehringer Mannheim #102 547) 90 ul 1 M MgSO4 Stock (150 mM MgSO4 ) add 225 ml distilled water (to 315 ml) Store at -20oC 100 mM Sodium orthovanadate* 1.839 g Sodium orthovanadate distilled water to 8 ml in a screw cap tube adjust pH to 10 if solution is yellow, place in boiling water until clear recheck pH, repeat as necessary adjust to final concentration by checking A265 nm, ext. coeff.= 2925M-1cm-1and adding distilled water as needed store at -20oC 3 M KI* 4.98 g KI add distilled water to 10 ml store at room temperature 0.5 M Na2 CO3 * 531 mg Na2 CO3 add distilled water to 10 ml store at room temperature L B Broth In 1 liter of ddH2 O, combine: 10 grams Bacto-tryptone 5 grams Bacto-yeast Extract 5 grams NaCl Stir until dissolved. |
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