quantify cDNA synthesis

John Thompson jrt at home.com
Tue Nov 7 19:42:45 EST 2000


Peter-Bram 't Hoen <p.hoen at lacdr.leidenuniv.nl> wrote:

>Dear netters,
>
>In an attempt to quantify the amount of cDNA synthesis from a certain
>amount of RNA in the absence or presence of different inhibitors, I
>thought it wise to perform the reverse transcriptase reaction in the
>presence of radiolabeled nucleotides ([32P]-alpha-dATP or
>[32P]-alpha-dCTP), then spot the cDNA on a Hybond-N membrane, cross-link
>by UV or heat, and quantify the spots with phosphor-imaging. However, I
>need to get rid off any free nucleotides. Will the nucleotides also
>adhere to Hybond-N? If yes, is there a method to wash the nucleotides
>away?
>
>Thanks for your answers,
>
>Peter

I used to use charged nylon membranes to quantitate incorporation in
DNA labeling reactions.  The nucleotides wash off nicely in 2-5X SSC
or SSPE.  Use a zero time point control as there will certainly be
some marginal signal from nucleotides alone, but as I recall the
nucleotides were >99% removed by simple washing.

Regards,
John Thompson






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