PCR of cDNA library

mikem at s-and-s.com mikem at s-and-s.com
Mon Apr 22 07:50:27 EST 1996


>   lazar at mail.med.upenn.edu (Mitch Lazar) writes:
>  Has anyone had success with PCR of a lambda cDNA library using one primer
>  hybridizing to a lambda arm with a second primer which is cDNA specific?  
My "take" on PCR of libraries is the following:

1.  It is not like PCR from RT-CDNA because in addition to the cDNAs, you have 
a huge amount of lambda DNA.  In addition, cDNA libraries usually have been
through 1 or 2 rounds of amplification, so some clones may be severely 
underepresented.  

2.  Design your cDNA probes so that they are not homologous to any portion of lambda.

3.  Do asymmetric PCR first with the oligo designed for the cDNA, to enrich the mixture for
cDNA specific ssDNA, then add the lambda oligo.

When I tried to do amplification from a library in this fashion, I was not successful, but
I believe it was because my particular clone was severely underepresented in the mix.
The above are some empirical guidlelines to get you started.
				
						Mike Morgan
						Technical Services
						Schleicher & Schuell

P.S.  We would also appreciate any references on a successful amplification from a cDNA 
library with only one specific primer, so that we can add it to our database.




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