Keith Savin savink at
Wed Jun 17 06:00:46 EST 1998

Ben Wielockx wrote:

> I used the Gibco-BRL kit starting from mRNA in an elution buffer. But I
> have no reverse transcription. Solution?
> Is there a method to measure the exact concentration of the obtained
> cDNA. (fenol extraction?)
> Any advise is welcome
> Thanks

I think the pH of the buffer for superscript RT is critical. Try
precipitating your RNA and redissolving it in DEPC-treated water before
adding it to your RT reaction. However, I've heard reports of problems
with some recent batches of this enzyme (maybe it's the buffer, not the
To measure the amount of cDNA you do a calculation based on the amount
of tracer 32P-dCTP incorporated and the amount of dNTPs present, etc
(it's in the manual). However, I just guess based on about 30-50% of the
amount of RNA I start with and use this as the basis for a few trial
ligations etc.

Keith Savin

Victorian Institute for Animal Science

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