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,)
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 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
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
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 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 dissolved.



*=As published in...
Waterman-Storer, C.M.  (In press)  Microtubule/organelle motility assays.  In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds.  John Wiley, NY.

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The Salmon Lab
University of North Carolina at Chapel Hill
Department of Biology
607 Fordham Hall,  CB#3280
Chapel Hill, NC  27599

To contact us:
Phone: (919) 962-2354
Fax: (919) 962-1625
Email: jccanman@email.unc.edu