Degenerate Primers

Wendy Bedale bedal001 at mc.duke.edu
Tue Apr 25 17:43:48 EST 1995


In article <3nj4lc$am5 at server.st.usm.edu>, jstemple at whale.st.usm.edu
(Jeffrey Shane Temple) wrote:

> I just had two 18 base primers designed for use in PCR.  They are
> degenerate in approximately 7 positions in that any nucleotide can pair.
> I was wondering if anyone with more experience knows what concentration of
> these primers to use in PCR?  It seems that you would use more than
> non-degenerate primers, but I am not sure how much more. Any help would be
> greatly appreciated.

I have been doing some PCR from bacterial genomic DNA with degenerate
primers.  My primers were 26-mers that were degenerate in 4 or 6
positions, and I used the same amount of them as I would in a normal PCR
(1 umole of molecules).  I had about 100 ng of genomic DNA.  The one thing
I did that was a little different was that I did 5 cycles in the beginning
with a very low annealing temperature (45 degrees C) and then did 30
additional cycles at 55 degrees C.  Hope this helps!



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