how to isolate ORF by PCR - (May/26/2008 )
I want to isolate ORF of AtSTP2 gene which is of 1116bp length.i designed primer by considering sequence from orf start codon to stop codon,but the problem is with the designed primers they are giving product size is of 250 to 370 bp long.I want to isolate whole ORF sequence of 1116bp size. how can i do this with pcr.please help me in considering points to design primer to get 1116bp ORF.
thanking you very much.
Hallo, it is probable your primers have low anellation temperature. IT should by 20-25bp(or longer 30bp) long, anelation temperature around 60 dg Celsius, GC contetnd 30- 80 percent. I dont know this gene is eucaryotic, but it can by alternatively spliced. But it is problem only if you work with mRNA, chromosomal DNA harboring full lenght. You can use Taq polymerase for long PCR template although I have amplyfied 1000 bp fgagment with common polymerase. Also you can prolong polymerization step 1 min for one kb.