I designed some real-time primers for pig using RefSeq sequences and Primer-BLAST program of NCBI. To check specificity, I BLASTed the obtained primes in most of the online available programs and got single product everytime.
I ordered the primers and surprisingly, when I check my primers with normal PCR, too many non-specific results were obtained. For confirmation, I set up real-time experiment and the melt curve also showed multiple peaks.
What possibly might have gone wrong with my primers? Please suggest.
I agree with the first two suggestions. What length primers are you using? Another option is to decrease the annealing time. On the real-time PCR, when do the non-specific products appear? If it is not until 40 cycles or so then you may be able to optimise but if they appear earlier then you need to design new primers.
Thank you for your suggestions. Primer length varies from 18-22 bp.
I have tried out with different temperatures. I guess I should work on Mgl2 conc. Also, I'll try out with annealing and extension time.
Check primer blast again with "nr" option (instead of ref seq), with max allowed mismatches. Also check the primers for Hairpin, self dimer and cross dimer. If all these are OK, then run an NTC and check on gel for any bands. You can also check NTC with Evagreen. With Evagreen what Ct you get for NTC?
What is your template? If it is RNA, did you do a DNase treatment to rule out genomic DNA contamination?
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