Variability Between Plasmid Preps in Transfections

Jonathan M Kaplan jmk19 at ciao.cc.columbia.edu
Sat Jul 30 18:03:21 EST 1994


I could use some help sorting out a problem, one that I am sure I am not alone
with.  I am doing promoter analysis of my favorite gene using wild type plasmidsas well as one with a mutation in the binding sites for my other favorite gene.
I would like to see if the mutation has an effect.  Initial experiments did shoe
(sorry) show an effect but upon multiple plasmid preps, I got whopping differences.  At one point, I did 4 wild type and 4 mutants together, doulble CsCl
banded, extracted together, dialyzed together against 5 changes of TE at 4 liters, spectrophotometrically quantified together, and transfected by CaPO4 
together into NIH 3T3 cells.  I still saw differences between plasmid the preps.

Q:  Are there other people out there who have experienced this and have you
    figured out the problem?  How did you solve it?  Would Quiagen columns
    help?

Thanks for taking the time to consider this problem, you can repost an 
answer or Email me at jmk19 at columbia.edu.

Bye.
Jon Kaplan



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