Removal of RNA from minipreps

Hanson Lab Lab_Mac_Hanson at QMrelay.mail.cornell.edu
Thu Mar 10 14:24:51 EST 1994


In article <1994Mar10.011914.1 at wkuvx2.wku.edu>, simmoaj at wkuvx1.wku.edu
(ALAN J. SIMMONS) wrote:

> Are there any reliable methods of removing RNA from alkaline minipreps
> of plasmid DNA?  We do not have an ultracentrifuge at our institution.
> The plasmid well serve as template in Promega's coupled TnT system
> using SP6, so I would like to avoid RNase. I've not had very good luck
> with NaOH hydrolysis of the RNA.  Any advice would be appreciated.
> -- 
> Alan J. Simmons			E-Mail: SIMMOAJ at WKUVX1.WKU.EDU
> Biology Dept.
> Western Kentucky University
> Bowling Green KY 42101

I have been using a PEG precipitation after RNase treatment of my
minipreps, and with one or two phenol extractions to get rid of residual
RNase I don't have problems doing in vitro transcriptions.  For the
reference to PEG precipitation check Maniatis or look in the Promega
catalog for their recommended midiprep procedure.  Original ref I think is
a Methods in Enzymology paper by John Lis from the early 80's.  You can
also separate DNAs by size with this method.  Only requires a mocrofuge!

Good Luck, 
Claudia Sutton, cas9 at cornell.edu 



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