5 replies to this topic

[obog360]

Hello

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?

thanks

[pcrman]

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.

[obog360]

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?

thanks

[pcrman]

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.

[obog360]

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.

[obog360]

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