Great signal but nothing on gel - (May/27/2008 )
We've been using the Qiagen RNeasy micro kit and Quantitect assays for small scale RNA isolation and real time PCR. We've noticed an unusual phenomenon. When we use the suggested 1:250 dilution, we've been seeing great amplification with CT values in the low 20s for our target and in the 18-19 range for our actin and microglobulin standards. Melt curves look clean. But then when we run it on a gel, there is no product visible. We've called tech support and they thought that the amplication we are getting is real but just not enough product to see on a gel (?). When we use more starting material, we can see the product on a gel but then we get no signal on the PCR. Anyone ever had this problem?
I know this problem. The reason for this is that some suppliers (Qiagen, ABI) use special modified nucleotides in their qPCR master mixes which can not be detected by EtBr on a gel or have excessive UNG in their masters so that the dUMP containing pcr products are digested right after PCR. Sometimes it helps to analyze the pcr product immediately after the end of the pcr run without letting it stand around for a while or immediately freezing it and analyzing it right after thawing. You won't have this problem with some other suppliers of qPCR masters. Hope this answers your questions.
By the way: I usually use the workflow reagents from Roche-Applied-Science (HighPure RNA Isolation Kit, Transcriptor 1st strand synthesis kit and FastStart Universal Probe Master (Rox)) for qRT-PCR TaqMan Probe assays. After using different suppliers for every single step I changed to one high-quality supplier for the whole workflow because of the great tech-support they offer (including competitive prices).
I know this problem. The reason for this is that some suppliers (Qiagen, ABI) use special modified nucleotides in their qPCR master mixes which can not be detected by EtBr
This solves a years long mystery why I wasn't able to watch my real-time products on EtBr gel while doing my thesis work (ABI machine and master mix) and now I just run it without problem (Roche machine and master).
Welcome. Nice that I could help you
i have a somewhat similar problem, in that i can see the reaction OK on the machine, but when i run the gel, only one of my primer sets gives a visible product!
Could it be the same reason as above? I´m not sure why one (visible) product would have such different nucleotide composition than the others (non-visible)...