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Tree_Truffle 11-11-2009 07:46 PM

trouble with designing degen primers
 
Ok folks, this is something I'm a bit unclear on.

I've been using CODEhop for designing my degenerate primers to clone a gene out of alligator.

Problem... I picked a gene that's small (~500bp) so there's not as many occurrences of conserved regions desirable enough to make more than one primer. At least that's what CODEhop tells me.

I've added the seq from Gallus, a few other aves, some mammals and xenopus for good measure of diversity.

Now I don't know the intricacies of codehop, but i'm assuming it takes into account potential hairpin loops or complementarity among the primer pair. Perhaps more so the former.

With the sequences in Clustal, I see multiple conserved regions, which vary from 4-5 amino acids with an occasional single aa, yet codehop won't capitalize on them.

I tried to copy and paste the conserved fragments and got some primers but are these usable? Any suggestions?


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