RT-PCR vs. conventional PCR - (May/11/2009 )
i have had great results in amplifying a gene of interest with RT-PCR (Taqman) but have not been able to do so with conventional PCR (used multiple primer pairs including the same primers used for RT-PCR). i am struggling to find an answer as to why this is happening. any suggestions or pointers? my amplicon length for RT-PCR is around 60 bp and the expected range for conventional PCR is from 60-900 bp. my Ct value is approximately 30 for RT-PCR.
is it possible that conventional td-pcr (20:20 cycles) is not sufficient? from what i understand, i should be able to amplify my gene using 20:20 cycles, correct? (even when my Ct for RT-PCR is 30)? ive also tried 20:30 cycles to no avail...
im perplexed as to why i can amplify with RT-PCR and not conventional PCR and im not sure where to go from here....
By RT-PCR are you meaning real time PCR (qPCR) or reverse transcription PCR (one step presumably if using Taqman)?
If you CT is 30, you should be able to see it after 40 cycles with conventional PCR, however, the fluorescent probes are much more sensitive than gels stained with EtBr or sybr-green/safe, so you still may not be able to see a band. If you are trying to resolve in an agarose gel, try using a polyacrylamide gel instead, it will work much better than agarose for this size product.