RT-PCR-Multiple bands-unusual problem

Olivier Gandrillon ogandril at cri.ens-lyon.fr
Wed Jun 26 03:21:48 EST 1996


Hi

My first thought would be to improve the specificity of your PCR 
conditions: try higher annealing temperature, less cycles and/or less 
template. Second thought: could the second band actually be primer 
dimers (they sometimes migrate quithe high)? Third: there exist some 
splicing variants in your tissue (less probable, I guess).


Paul Mozdziak wrote:
> 
> I'm doing some RT-PCR to see if a particular factor is expressed in
> muscle. First I am using previously published primers. Second my
> positive control is a small amount of a cDNA probe for my factor of
> interest. The probe samples amplify beautifully. However, when I run
> my rt-pcr stuff from muscle out on a 3% gel (sigma agarose (50-1000
> bp) I get two bands (one at 150 bp--what I should get and one a few base
> pairs smaller)--they just barely resolve. I ran these samples out
> shortly after I finished the pcr.
> 
> Any ideas to explain my problem and too solve it?
> 
> My RNA was not DNAsed--and it's just total RNA. There was alot of RNA at
> the beginning of the procedure in my muscle samples ~1 microgram.
> 
> Thanks much
> 
> Paul

-- 
Olivier Gandrillon AKA ogandril at cri.ens-lyon.fr

Meet me at http://www.ens-lyon.fr:80/~ogandril

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