Protocol Online logo
Top : Forum Archives: : Molecular Cloning

About Reverse transcriptase PCR followed by PCR - primers (Mar/20/2007 )

Hi, can you help me
can I used the same oligo dT for the RT and the following PCR.
At which temperature should I run it....because the Tm for the oligo dT and for the other primer is different.

thank you


I wouldn't recommend it because you are going to get a lot of secondary bands. If you think about it, your PCR will only be specific for the forward primer only, anywhere it binds on the antisense strand a product will be produced. To answer your question, it's pretty simple really, just think about the RT-PCR. It can't happen if the oligo-dT isn't annealed to the template right? So whatever temperature you run the RT-PCR at will be fine. A touchdown PCR might be good to reduce secondary bands.