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RT PCR - dilution prior to PCR (Sep/02/2004 )

Hi
I wonder could you answer a basic question for me: Having performed 1st strand synthesis do I have to dilute the cDNA prior to PCR analysis. I designed primers and am using PCR to amplify the product before electrophoresis but I am confused as to whether I need to dilute the cDNA prior to performing the PCR reation. In the promega protocol it says to dilute to 100ul but in others it doesn't mention it.

thanks
Dtkk

-drdtkk-

If your target is a rare message, I would not dilute the cDNA before PCR, however if it is really abundant...diluting would be better for the PCR reaction. If you have enough sample I would try both ways and see which one gives you the best yield.

-phegene-

I agree.

I didn't do dilution until I found the protocol coming with the promega kit, then I think diluting RT products is good in two ways: giving you more templates and making detecting subtle expression changes easier.

-mario2004-