b-gal assay

the End jgraham at bronze.ucs.indiana.edu
Mon Sep 11 21:16:04 EST 1995


Be sure that you have the proper concentration of SDS, I 
think it is 0.1% or 1/10 of a percent. Chloroform should
be relatively fresh.

I believe the SDS/chloroform is more of an "in situ" assay
relative to the classic toluene. 

I should really go ask Art Koch himself. :)

Jim
J. Graham
Biology Department

PS. Despite Miller's contention, this assay on E. coli is not 
as simple and reproducible as suggested. I've found that the 
amount of time that cell cultures are kept on ice can have 
dramatic effects on the activity assay. Try to have percisely 
15 minutes for every culture, and return to ice for cell OD assay
after rather than prior to the hydrolysis assay.

I also found that cells must be thoroughly vortexed upon dilution,
and following addition of SDS prior to addition of chloroform
(20 ul ea./ ml) for reproducible replicate samples, and even then
there were bad days. These extra steps are a significant amount 



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