Is nested PCR always neccisary? - (Apr/24/2008 )
I just a project using BSP and have a question about whether I need to use a nested PCR. I am looking at three different CpG islands with amplicons from 280 to 440 bp. For two of the targets I have single bands in my gel following the first round of PCR. One is very strong band whereas the other is fainter but still the only band present. The third target I have five distinct band with the largest band being the proper size. I know that for the last target Im going to have to do the nested PCR but what about the other two. Does it help with the sequencing to do the nested PCR or am I just wasting time? Thank you in advance to everyone. This forum has been invaluable.
I sequenced a gel purified band directly with no nested PCR reaction and it worked very well. I'm not even sure you need a nested PCR with your third target, if you can get sufficient DNA from the band you have.
I made similar experiences too. I designed a nested approach for a BSP, however the extern primer didn't work very well but the intern primer were ok. So I amplified two times with the intern primer to get enough product. The PCR was specific. However this does not always work, the most of the times I need a nested approach to get specific products.
To obtain sufficient products for sequencing, we usually just reamplify the first round PCR product using the same set of primers. We found two rounds of PCR are always necessary. Of course, nested PCR with an inner set of primers is an alternative. We prefer the former because it is simple. For those non-specific bands, try raise the annealing temperature or use Hot-start techniques or enzymes.