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
>Thanks for your answers,

Try TCA pptation or DEAE filter paper as per Maniatis.

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

More information about the Methods mailing list