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trouble shooting RT-PCR

Quimica proteios at indirect.com
Sat Apr 15 23:36:49 EST 1995


In article <LRM-1404951353010001 at thunderweasel.ahabs.wisc.edu>,
LRM at zeus.ahabs.wisc.edu (Lee R. Martin) wrote:

> 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
Im just listening in here (hope you dont mind). I would really appreciate
it if when you send the protocol, if you could just tack on a copy and
send it to me (proteios at indirect.com). This method sounds very
interesting. Has anyone attempted to use this method with respect to
plasmids?



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