Bands seen with qPCR missing in regular PCR - (Aug/31/2011 )
I am trying to detect a gene that is expressed at a low level. I do RT to generate cDNA and then I use this cDNA for qPCR and for standard PCR. I use the same primers for both qPCR and for regular PCR. After doing qPCR and getting cycle numbers I just ran the products on the gel to make sure I was getting bands, I saw 2 bands one 200 and one 700, they were of the same intensity. However, after running the standard PCR on a gel I just see the 200 band. For the standard PCR I used jumpstart taq with a 1:00 elongation time at 72. Does anyone know why this is happening?
What is the 700 bp band -- DNA contamination? Is everything the same except the taq in the two reaction? Many things such as template and primer concentration, taq, could affect amplification efficiency especially the bigger product.
Hey thanks for responding. The RNA was treated with DNase before qPCR and PCR so I don't think it is DNA concentration. No the primer concentrations are different I used 100 nM for qPCR and 250 nM for regular. I also used .3 ul of cDNA template for qPCR and .5 ul of cDNA template. The melting temperature was the same for both. I just tried PCR with different melting temperatures from 50 to 60 but still no band. I am using jumpstart taq for the regular pcr, do you think I should try with fidelity? Would jumpstart have a hard time amplifying 700 bp?
Are both the 200 and 700 bp bands expected by you? They are two isoforms to the same gene? JumpStart is a very good taq for amplifying difficult templates. The size is not a problem at all for JumpStart.
yeah the gene I'm looking at has a gfp reporter. The band I would expect based on gene sequence is 200 bp. But I think one band is the normal gene and the other may be the gfp knockin. I think GFP sequence is around 500 bp so it would make sense to have a 700 bp band.
never mind I think you were right it must have been DNA contamination because I am no longer getting the band with qpcr either. thanks