quantify cDNA synthesis

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Nov 7 10:48:02 EST 2000


In article <3A081274.37949F8 at lacdr.leidenuniv.nl>, the eminent Peter-
Bram 't Hoen at R.U. Leiden 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
>

Try TCA pptation or DEAE filter paper as per Maniatis.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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