PCR specificity problem - (Sep/30/2004 )
I am having a nightmare with a PCR I am trying to do on the CYP11B1 promoter. I have been trying to amplify approx 2kb of it for what seems like forever now. I went from no bands to eventually having a band after careful optimization. It then worked for a few attempts but now I am getting a smaller second band as well!!! Is it possible that primer degredation could be causing this? I have tried altering Mg and primer concs but cant seem to get rid of it completely. Any ideas?
Primer degradation is unlikely. How small is the smaller product? Could it be primer dimer?
Hey - its not primer dimer, it seems to be about 300bp. I guess I should try altering the cycling temperatures slightly - im just reluctant to do so as it took so long to get a band in the first place so I was just hoping it could be something else.
The small band you got could be amplified from another genomic region other than the one of your interest. It is not a surprise to get non-specific amplification with any primers since in a genome, there are many paralogs and duplications. You can check if your primers anneal to some other places using the in silico PCR tool at http://genome.ucsc.edu/cgi-bin/hgPcr?db=hg17.
Bioline make an accurate PCR mix called BioXact. It comes with a high specificity additive which completely removed the non-specific bands from my reaction.
I did my cloning, picked a couple of plasmids for sequencing and both were correct.
They also make one for long targets.
have u tried reamplifying ur PCR product under same conditions?
if not try doing this either after band eluting ur required product or using the previous PCR mix as template