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cDNA PCR on RNA virus

Lee R. Martin LRM at zeus.ahabs.wisc.edu
Sat Apr 15 19:24:40 EST 1995

In article <m0rwobE-0004Z6C at uctmail.uct.ac.za>, ed at molbiol.uct.ac.za wrote:

> I know there was some discussion on this...we want to PCR about 2.2 
> kb of a cucumber mosaic virus RNA, and although we can do smaller 
> pieces no prob, the bigger eludes us.
> Any hints on good cDNA PCR for longer products?
> Which enzymes, what conditions?
> Ta in advance,

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

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

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

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