BLAST is giving me a result totally unrelated to the gene I expect - (Apr/28/2013 )
After I obtained the results from sequencing, I proceeded to curate the sequences and identifying (with Vector NTI) the primers I used in the sequence. The primers, although are degenerated, were found with no mismatches.
The problem is that when I blast the squence using BLASTn or BLASTX, the results are malate synthase, a gene/protein that I totally didn't expected and totally unrelated to the gene I'm interesed. These results were like 13 of the 20 sequences while 5 were of the gene I was expecting.
Please tell me what went wrong. I'm a bit desperate. I used a clone library with pGEM and chemocompetent DH5-alpha.
What were the BLAST scores?
For the malate synthase:
Select seq gb|CP001874.1|
Select seq gb|CP000613.2|
Select seq gb|CP003382.1|
For Roseiflexus hypothetic protein
Select seq gb|CP000686.1|
Select seq gb|CP000804.1|
Unless I discovered the Sox system in a Chloroflexi member, I don't know why does this hapen.
You said primers were degenerated, how much?
And, you seem to be "fishing" for some unknown gene, maybe you degerated primers so much, they only caught something very different.
You probably won't find any mismatch in primers, when only certain sequence of degenerates binded the target. It's unclear if you knew the exact sequence of the primer binding site of your gene, but probably not, because you woudn't use degenerate primers then.
I think generaly checking specifity of degerate primers is difficult, when you degenerate more than one base for example. I wouldn't consider it that much impossible to amplify something else than you wanted, with such primers.