Help with DE-81 paper

[Arthur Hunt]

In article <2qm7kr$igf at louie.udel.edu>
landel at helios (Carlisle Landel) writes:
 
>
>
>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
>
 
We use a similar approach for quantitating polyA polymerase.  I don't know wher
e the important differences may lie, so I will give you a brief summary of what
 we do:  stop reactions (phenol/chloroform), spot aqeous phase onto DE-81 paper
, wash with 4 changes of 5% Na2HPO4, once with water, then count.  We dont dry
the filters before washing with phosphate.  Typically, if 1 microcurie of count
s are spotted, background ends up between 200 and 500 cpm.
 
Good luck
Arthur Hunt
Dept. of Agronomy
University of Kentucky