RT-PCR cloning: oligo dT-primed cDNA synthesis or anchored? - (Feb/08/2008 )
A quick question for those with more cloning experience than I.
I've decided to try and amplify a 1.5 kb sequence from mRNA to clone into an expression vector. However, in our lab we can't agree on how to make the cDNA. I think I should prime cDNA synthesis using a gene-specific reverse primer, but my colleague seems to think we should do RT with oligo-dT, and only use gene-specific primers in the PCR step. I say this might be necessary if we started with total RNA, but because we're using mRNA, gene specific primers should suffice.
Who is right???
Thanks in advance for your help,
Either should work well.