Large-scale preparations of plasmid DNA, what wrong?
Thomas H. Rude
rude0001 at acpub.duke.edu
Fri Mar 18 20:39:18 EST 1994
|In article <2mch5d$815 at klaava.helsinki.fi>,
|Huanquan Zheng <zheng at cc.Helsinki.FI> wrote:
|>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.
When we have performed CsCl purification.
1. You need to dilute the CsCl by 1:10
this is required to get the salt conc. low.
(ex. 100ul of Cscl to 900ul of H2O)
2. Then you need to add 2 vol. of 100% ETOH or
1 vol. of Isopropanol.
** One of the problems may be that there is also too
large of a volume. If you have saved everything, you
may want to reduce volume and try reprecipitation of
Duke Medical Center
|>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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