PCR with degenerate primers

Pat thoyts at bsa.bris.ac.uk
Fri Jul 30 15:31:11 EST 1993


>I would like to know what parameters should I change in order to
>improve the possibility of identifying a specific gene by using
>degenerate primers ??
>
>I have lowered the annealing temperature to give the primers more
>chance to anneal but I would like to hear some advice from the
>experts on PCR, and specifically those who have used it to identify
>new genes from total DNA. Thanks very much in advance for your help.
>
>                            N. Salazar
Well I'm no expert, but I recently used some degenerate primers on an 
aliquot of a lambda cDNA library with success. I used a LOT of primer.  I 
think for 6 50ul reactions I used 1 ul of undiluted primer (probably around 
400 ug/ml - ie :as you get from the sysnthersiser). I also upped the 
magnesium to 2.5mM Otherwise, as normal PCR.  I think using a lot of primer 
helps a lot.
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Pat Thoyts                                           thoyts at bsa.bris.ac.uk
                                                     Farell at DF & HarpersTale
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