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sucrose gradient protocol


A Dohlman Lab Protocol

Sucrose Gradient Protocol

from Song et al. 1996, Modified by Ginger Hoffman

 

FROM HERE ON OUT KEEP EVERYTHING AT 4oC

RUNNING GEL

SOLUTIONS

S/K (100 ml):

1.2 M sorbitol (21.84 g/100ml)
0.1 M KPO4 pH 7.5 (9.5 g dibasic (fw = 268) 1.99 g monobasic (fw = 136)

filter sterilize!

zymolyase buffer:

to S/K buffer add:
2 ul/ml of beta mercaptoethanol
20 ul/ml of zymolyase (stock solution 5 mg/ml in S/K)

spin before using!

Lysis buffer C:

20 mM TEA pH 8
0.8 M sucrose
1 mM EDTA
1 mM DTT (add fresh)
1 mM AEBSF (add fresh)
10 ug/ml leupeptin (add fresh)
10 ug/ml pepstatin (add fresh)
10 ug/ml benzamidine (add fresh)

mix first 3 ingredients and filter sterilize

Sucrose:

60 g sucrose + 5 ml 200 mM TEA + H2O to 100ml for 60% solution
also prepare 50%, 40%, 30% (all also dissolved in TEA)

filter sterilize

TEA buffer:

triethanolamine 0.2 M stock; filter sterilize


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