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
	   the prep.  

	Good luck!

	Tom Rude
	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? 
|>
|>
|>HQ





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