Sequencing with degenerate primers
Peter J. Neame
pneame at com1.med.usf.edu
Fri Nov 25 15:02:44 EST 1994
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.
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.
SHCC and USF, Tampa
pneame at med1.com.usf.edu
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