Sequencing with degenerate primers

[Peter J. Neame]

Eric Lippay (lippay at vaxd.gat.com) wrote:
: 		I would like to hear from people who have used degenerate primers
: successfully for sequencing.  Any hints or good advice to make it work
: would be appreciated.

Hi Eric!

We've had some success using primers that are about 60- fold degenerate 
for sequencing PCR products in shark genomic DNA. It is absolutely 
critical that the PCR product be gel purified; use the highest resolution 
you can manage. Even better is to "nest" the degenerate sequencing 
primer, if you have enough data to do this. 

As to the actual sequencing, I am a bit more fuzzy about this, as we send 
ours out to a core lab. That rather indicates to me that you don't need 
to do much more than regular cycle sequencing. 

Good luck, and best wishes.

Peter Neame
SHCC and USF, Tampa
pneame at med1.com.usf.edu