Help with DE-81 paper

Carlisle Landel landel at helios
Mon May 9 15:49:31 EST 1994


Hi!

We were doing an assay of our terminal transferase, where we spiked
the reaction with a little 32P dATP.  We decided to measure our results
by using DE-81 paper to trap the reaction product, as outlined in Maniatis.
Basically, you spot some of the reaction on the paper, let it dry, then 
plop it into some 0.5M Na2HPO4 to wash it, then wash it with EtOH and
dry it and count it.

We had it set up that we expected about 600 dpm/residue added.

The problem is that we get real high background: >7000 dpm at our 0
time point, and no significant rise in that value with time.  We figure
that either the reaction didn't work or that we have so much background
that we can't tell if it *is* working. 

So my question is this:  is there some way to knock the background down
to near-zero with DE-81?  

Otherwise, we are going to run the reaction products on a gel and then
count the band.  (We are avoiding precipitation because we want near
100% recovery for quantitative purposes.)

Thanks for the help.

Regards,

Carlisle Landel




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