DNA Extraction for PCR.

wetsel_r at msdisk.wustl.edu wetsel_r at msdisk.wustl.edu
Mon Oct 11 13:54:58 EST 1993


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



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