RT-PCR - 2 kb fragment in RT-PCR (Jan/03/2005 )
I am using SuperScript First-Strand Synthesis System for RT-PCR. I performed the reaction with control RNA and it worked. I tried 4 different primer sets for 4 different genes. 3 of the primer sets which amplify smaller than 500 bp fragments are working but 1 primer set which should amplify a 2 kb fragment is not working. Are there anyone who faced with problems with moderately long product amplifications?
How long did you run your RT? The RT extension should run at 42C for at least 1 hour. Using random primer instead of oligo dT may help. But if other primers work with your cDNA, the problem may not be at the RT step. Make sure your primers are good and PCR conditions are optimized for the 2kb product.
according to your descrition that short fragments can be amplified but long one can not,I suppose it should ascribe to the unsatisfied RT reaction rather than the follwing PCR. anybody more suggestions ?