degenerate primer question

Stephen R Fox sf2w+ at
Sun Aug 4 18:11:45 EST 1996


I have generated 2 degenerate primers for a drosophila gene I am working
with. I plan to use them to find and clone a vertebrate homologue of the
gene. Unfortunately, my primer control reaction has not worked. The
control consists of using the primers to PCR amplify an approx. 300bp
region of cDNA containing the Drosophila gene of interest. I have tried
low annealing temps, tried starting with very low annealing temps for a
few cycles and then increasing the annealing temp, I have tried varying
concentrations of reagents, but still have had no success. I always run
a second control on the same cDNA using non-degenerate primers and this
always works. Can anyone give me a good protocol for getting degenerate
primers to work, or perhaps point me towards some good literature? All
help and advice would be greatly appreciated. Thanks!


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