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常用缓冲溶液的配制
2010-09-29
来源：亚科官网
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 Na2HPO4*7 H2O ,0.545 g KH2PO4 ,pH 7.2
Mounting Media :20mM Tris pH 8.0 ,0.5% N-propyl gallate ,90% Glycerol ,Store at 4oC
PM Buffer* :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,) ,
PBS (5x in 500 mls) :20.45 g NaCl ,0.465 g KCl ,10.142 g Na2HPO4*7 H2O ,0.545 g KH2PO4 ,pH 7.2
Mounting Media :20mM Tris pH 8.0 ,0.5% N-propyl gallate ,90% Glycerol ,Store at 4oC
PM Buffer* :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) ,
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) ,
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 KH2PO4 (1.6 mM KH2PO4) ,
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 KH2PO4 (1.6 mM KH2PO4) ,
2.15 g Na2HPO4 (15 mM Na2HPO4) ,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 = 7.0 Stock* :119.15 g HEPES (free acid) ,distilled water to 400 ml ,
1M MgSO4 Stock* :24.074 g MgSO4 ,distilled water to 200 ml ,store at room temperature
1 M HEPES, pH = 7.0 Stock* :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 = 6.9 Stock* :151.2 g PIPES (free acid) ,distilled water to 400 ml ,
distilled water to 500 ml ,sterile filter and store at 4oC
1 M PIPES, pH = 6.9 Stock* :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 ,
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 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
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),
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 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
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 Na2CO3* :531 mg Na2CO3 ,add distilled water to 10 ml ,store at room temperature
L B Broth:In 1 liter of ddH2O, combine:10 grams Bacto-tryptone,5 grams Bacto-yeast Extract,5 grams NaCl,Stir until dissolve
