Large-scale preparations of plasmid DNA, what wrong?

Huanquan Zheng zheng at cc.Helsinki.FI
Fri Mar 18 10:25:33 EST 1994

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.		

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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