RT-PCR smear with oligoT

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hftuan
hftuan's picture
RT-PCR smear with oligoT

Hi,

I am trying to map the 3' end of a bacterial transcript and to do that I first added a bunch of As to the 3' end of the isolated total mRNA using poly(A) polymerase, then I did a RT using oligo(T) primer to synthesize first strand cDNAs. Now to specifically fish out the cDNA of my target transcript, I continued with PCR using, again, oligo(T) as a primer along with another specific primer. I know my RT-PCR smear came from the annealing temperature that is too low, but oligo(T) requires a low annealing temp. Now I am not sure what to do. I am thinking I could isolate the region of the smear where I think my target is, and do PCR using oligo(T) with a new nested primer, but I don't think that'll necessarily resolve the smearing problem if I keep the temperature as low as before. I also looked into the possibility of modifying a touchdown PCR protocol so the annealing temperature go gradually from my nested primer (which is around 50) to the annealing temperature of oligo(T) (around 30). Would that possibly work??

I would appreciate any suggestion!!! Thank you!!