PCR specificity problem
Posted 30 September 2004 - 04:11 AM
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?
Posted 01 October 2004 - 01:36 PM
Posted 03 October 2004 - 10:32 AM
Posted 03 October 2004 - 03:51 PM
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.e...n/hgPcr?db=hg17.
Posted 06 October 2004 - 05:34 AM
I did my cloning, picked a couple of plasmids for sequencing and both were correct.
They also make one for long targets.
Posted 06 November 2004 - 06:22 AM
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