quantify cDNA synthesis

Peter-Bram 't Hoen p.hoen at lacdr.leidenuniv.nl
Tue Nov 7 09:32:21 EST 2000


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






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