trouble shooting RT-PCR

Lee R. Martin LRM at zeus.ahabs.wisc.edu
Fri Apr 14 14:53:01 EST 1995


I have two tips that might allow you to amplify your vRNA:

1) Purify the virus prep before vRNA extraction. I recommend purification
by sucrose pellet.

2) phenol/chloroform extract the cDNA after the RT reaction and before PCR
amplification.

I regularly do RT-PCR on picornaviral RNA, and have had to totally
trouble-shoot the system. By doing 1 &/or 2 I've been able to RT-PCR up to
3.5 kB of vRNA from picornaviruses, when w/o these steps I'd get no PCR
products (or crud at the bottom of the gel). If you need protocols, I'll
be happy to send them your way.

Good luck!

Lee Martin

-- 
Lee R. Martin
LRM at zeus.ahabs.wisc.edu



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