常用缓冲液配制－2

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
 
PM  Buffeundefined
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

Valaundefined
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 Systeundefined
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 Buffeundefined
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 Buffeundefined
 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 Buffeundefined
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 Buffeundefined
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 Buffeundefined
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

PBundefined
 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   Stocundefined 
24.074 g MgSO₄
distilled water to 200 ml
store at room temperature

1 M HEPES, pH = 7.0  Stocundefined
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  Stocundefined
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 Stocundefined
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 Stocundefined
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~undefined
1.542 g dithiothreitol
distilled water to 10 ml
disburse into 500 ml aliquots and store at -20oC

1 M KCl Stocundefined
 74.55 g KCl
 distilled water to 1 liter
 store at room temperature

10 M NaOH Stocundefined
 40 g NaOH
 distilled water to 100 ml
 store at room temperature

100 mM MgGTP Stocundefined
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 Stocundefined
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 Stocundefined
292.2 g NaCl
distilled water to 1 liter
store at room temperature

3.6 mM Brefeldin undefined
10 mg Brefeldin A (available from Epicenter Technologies Madison, WI)
absolute ethanol to 12 ml
store at -20oC

30 x Naundefined
 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-undefined
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-PNundefined
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 orthovanadatundefined
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 Kundefined
 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.