reverse transcription PCR - (Jan/09/2007 )
I will do reverse transcription PCR. Now I am using perlprimer to design my primers. I have following questions.
1) should I paste the mRNA or DNA sequence into this software?
2) what is the general size of the product? 800bp is too large?
3) if i input the mRNA sequence, how can i know that oen primer should cover the intron region or intron-exon junction region?
I am confused. I hope you can help me.
Thanks a lot.
I haven't used perlprimer, but...
2) Try to keep it under 500. I usually have products between 100 and 300.
3) Check it out manually using PubMed et al. You should have the sequence if the introns, and when you map where the primers are going to be, you'll have you're anwer. In my experience, exons have been pretty short, and it's quite easy to have primers that extend over several exons.