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
>
>-- 
>--------------------------------------------------------------
>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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