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John Stiles stiles at uhunix.uhcc.Hawaii.Edu
Wed Jun 15 14:10:11 EST 1994


In article <Pine.3.87.9406142051.B24186-0100000 at csuvax1> obrien at CSUVAX1.MURDOCH.EDU.AU (Philip OBrien) writes:
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>Date: Tue, 14 Jun 1994 19:45:50 +0800 (WST)
>From: Philip OBrien <obrien at csuvax1>
>Subject: DEGENERATE PCR
>To: methods-and at csuvax1
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>Dear netters
>In order to clone pectic enzyme genes from fungi we have identified 
>conserved regions within the genes (from amino acid sequences)and want to 
>make PCR primers from these regions.  However we have to cope with 
>2-4fold degeneracy at each codon (there are 6 codons/conserved region).  
>Although we can make the primers how does this level of degeneracy affect 
>the efficiency of PCR?  Are we likely to get to many non specific 
>products?  I would be interested to hear from anyone with experience of 
>this method.
>
>Phil O'Brien
>BES, Murdoch University,
>Murdoch WA 6150 AUSTRALIA
>email: obrien at csuvax1.murdoch.edu.au
>
Phil,

	We've done a bit of this and the rule of thumb seems to be keep the degeneracy to about 512 fold for best results. I have to admitt that I have violated this a bet and it still worked.

John Stiles
stiles at uhunix.uhcc.hawaii.edu




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