rt-pcr problems

Jody K. Hirsh jkh141 at nwu.edu
Wed Jan 3 13:46:20 EST 1996

In article <4c9vuv$6f0 at neuro.usc.edu>, william at neuro.usc.edu says...
>Hello all,
>I have been trying to amplify messages using RT-PCR, but I don't get any
>bands after the PCR.  I failed to see bands even with primers from 
>Clontech designed to amplify G3PDH.  The protocol that came with the
>primers recommended that I use oligo-dT or random primers during the RT
>step, but I just used the 3' PCR primer.  Has anyone out there have
>success with RT-PCR without using oligo-dT or random priming?  I don't 
>understand why random or oligo-dT priming would give better results.
>William Sun, Ph.D.                      Phone: (213)740-3406
>Hedco Neuroscience Building             Fax:   (213)740-5687
>University of Southern California       Pager: (310)499-8670

Just to be on the safe side, why don't you try using the oligo dT or random 
primers, and also use the control also that comes with the Superscript kit. I 
highly suspect that  your trouble is degraded RNA!!! My exprerience is that 
you need to use guanidinium to get good RNA; I highly recommend Trizol.  
Also, did you run a formaldehyde gel on your RNA to see if it was any 
good? G3PDH is very highly abundant so this sounds very suspicious.
Jody K. Hirsh
Northwestern University, Chicago, IL.   USA
jkh141 at nwu.edu

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