DNA Extraction for PCR.

Leonard N. Bloksberg bloksber at pilot.msu.edu
Mon Oct 18 11:32:00 EST 1993


In Article <11OCT93.13545881 at msdisk.wustl.edu> "wetsel_r at msdisk.wustl.edu" says:
> In a previous article, Yves Brun <ybrun at bio.indiana.edu> wrote:
> >>Greetings Netters:
> >>
> >>I have a quick question for you:  What reference do you use, or protocol 
> >>do you use for the extraction of genomic DNA for use in PCR?  I'm not 
> >>looking for something that would produce DNA of such quality for genomic 
> >>Southerns, we have those protocols and they take 2.5 days!  I'm
> >basically 
> >>looking for something quick, easy, and reproducible where I can spend
> >more 
> >>time thinking about the PCR reaction than the DNA extraction!
> > 
> >	I don't know what your source of DNA is but this is what I use for
> >bacterial genomic DNA (Caulobacter crescentus, a Gram-negative). It is
> >fast and gives very high quality DNA.
> 
> Yves:  Thanks for the reply... my mistake.  I'm looking for 
> a protocol for eukaryotic tissue, specifically tissue culture
> cells. 
> 
> I'm using it as a check to see if stably transfected cDNAs
> within an expression vector are present in some form or another. 
> So my goal is to take a small sample of the stable transfectant, shortly 
> after removal of G418, and test them to see if the contruct is present 
> prior to expanding, sublcloning, and using much labor and $$$.
> 
> I have one transfectant which seemingly has kicked out the cDNA but 
> retained G418 resistance as determined by immunoprecipitation and southern 
> analysis.  So basically, I'd like to check my other transfectants via PCR.
> 
> Thanks again,
> David
> haviland at kids.wustl.edu
> 
> 
> 
> 
When you say eukaryotic cells, do you mean plant or animal?  The DNA
extraction procedures differ significantly.  
.
.



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