RT-PCR and gels

walter wlech at medicine.medsch.ucla.edu
Sat Jul 29 15:22:14 EST 1995


betts at ORION.ETSU.EDU (GORDON BETTS) wrote:
>I believe it is possible to run RT then take 3 ul or so and run
>on a 1% agarose gel to verify you have a product. Correct?
>If not, then how can you check to see if your RT worked before 
>wasting reagents on PCR?
>If you can run your RT product on a gel to confirm cDNA is present,
>then could you suggest a procedure that is reliable? Mine's not.

Depends on expected yield of your RT rxn. Perhaps there is cDNA there, 
but at concentrations lower than the detectable limit of EtBr/agarose. 
Try using a labelled RT primer and running a polyacrylamide gel>autorad 
to verify product. I don't do this myself ($$); usually I run a positive 
control with DNA at a copy number equal to (or slightly lower) than the 
expected copy number of my cDNA product. If the positive control works 
than I can assume the efficiency of my RT rxn needs to be improved.





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