Large-scale preparations of plasmid DNA, what wrong?
smithwhi at students.msu.edu
Fri Mar 18 20:23:00 EST 1994
In Article <2mch5d$815 at klaava.Helsinki.FI> "zheng at cc.Helsinki.FI (Huanquan Zheng)" says:
> I am currently doing LS prep of Plasmid DNA, the following
> are what I did, But I can not get any plasmid DNA. I do not
> know what wrong.
> Cells were cultured LB/Amp no problem
> Lysis by Alkali no problem
> Purification of Plasmid DNA by CsCl EtBr Gradients no problem
> Removal of Et-Br with butanol no problem
> remove CsCl I was not sure if it was OK, I did it as:
> 1.4 ml aqueous phase
> 2 ml miniQ water
> 18 ml 70% ethanol at 4 C 15 min.
Why not dialyze the DNA? Yes I know it is old fashioned, but it works.
> finally, After centrifugation (8500 rpm 30 min. 4 C), I did not
> get any DNA pellet to dissovle in TE.
> If you do LS-preps of plasmid DNA (pBS and pET22b), what is
> the best way?
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