Degenerate PCR - Sequencing?

Margaret M Lanterman djdlab at bimcore.emory.edu
Sun Mar 13 14:39:55 EST 1994


In article 2D7B7997 at QUCDN.QueensU.CA, GERLACHJ at QUCDN.QueensU.CA (Jim Gerlach) writes:
>We have been doing PCR with degenerate primers (17mers, usually 256 fold) 
>and obtaining products in the 150-300 bp size.

>I'm wondering what are the chances of doing sequencing using the 
>degenerate primers. Anyone tried this (or some other nice trick)? Thanx for 
>any sugguestions or help.

Well I am doing the same thing.  One product was 600bp and I was able to direct sequence with sequenase, using my degenerate primers.  Another product was 300bp and I also got some sequence although not as good.  Anyways, it gave me enough sequence to know if it was what I wanted or not (in other words it told me I shouldn't waste my time cloning it because it was a gene someone else has cloned).
I am still trying to find my gene of interest.  My most recent products are smaller (200bp) and I am also wondering if this is too small to direct sequence with my degenerate primers.
Margaret M Lanterman






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