RT-PCR gene specific amplification problem - (May/09/2007 )
okey.. I've been trying to amplify this fusion and hn gene from Newcastle disease virus (NDV V4 (UPM)) for sometimes now but no success there. I've design the primer accordingly to the gene sequence, did gradient PCR, and tried with different amounts of starting template. No band at all. I'm out of idea on how to troubleshoot this problem. I did BLAST of this primers, and comes out as 100% match. So why isn't there any amplification? Please advice...
FYI, no primers have been designed and published for amplification of full length gene from this strain. I've tried using the primers of similar strains, but no product. My viral template is ok, as i've done denaturing agarose, and is able to be amplified with the use of primers targeting the gene cleavage site (not for full length). And the primers I have designed are able to amplify NDV strain AF2240. Please help!
Wht is your result with Positive control? wht is the GC content and Tm of your primer? wht annealing temp. you have tried.
Run ur PCR product on 6% native continous PAGE. if there is very very small product was formed still ur able to see that. all the best.