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Quantitating Radioactive label in DNA and RNA

Nicola Fidler bt048478 at dingo.cc.uq.oz.au
Tue Jul 5 00:13:38 EST 1994


Stephen_Lasky at brown.edu (Stephen R. Lasky) writes:

>In article <2us9ns$eso at matt.ksu.ksu.edu>, hlhinkel at ksu.ksu.edu (Heidi
>Lanora Hinkel) wrote:

>> 	I am looking for methods with which to quanitate the
>> amount of P32 -ATP incorperated by end-labeling with T4 kinase,
>> in both DNA and RNA probes. I would like to hear of any methods
>> that you know of and which one you think is best. Thanks in
>> advance
>> 		Heidi Hinkel
>>

>The easiest ways are to separate the incoroporated label from the
>unincorporated 32P and then just count 1 microliter in liquid scintillation
>coctail.  

>You can separate the incorp from unincorp many ways.  The old way was to
>make your own G-25 cols. in 5 ml plastic pipettes and wash the label
>through, monitoring with a geiger counter.  Collect the first hot fraction
>and count an aliquot.  The newer ways include using G-25 spun columns (like
>from B-M) (I used to spin through 2 cols if I really needed accurate
>quantitation but that's probably not necessary) or you can use push columns
>like Promega's nuctrap.  I think that's the easiest and fastest way  and it
>keeps the label very concentrated, then just count 1 microliter.


>There are other ways if you dont want to separate the incorp from unincorp.
> They all involve precipitating (TCA) or binding (p81? paper) the labeled
>macromolecules and washing away the unincorporated label with either lower
>concentrations of TCA of phosphate buffers.  All of the MolecBiol manuals
>(like Maniatis) have good descriptions of how to do this.

>Hope that helps.

>SRL
>-- 
>***************************************************************
>Stephen R. Lasky, Ph.D.       Brown University/Roger 
Williams Medical >Center
>e-mail: Stephen_Lasky at brown.edu         LandLine: 401-456-6572
>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>America may be unique in being a country which has leapt from barbarism to
>decadence without touching civilization:  John O'Hara
>***************************************************************
Even easier is to count 1 ul in water ie Cerenkov radiation. You can 
either get an absolute measure by also counting a known amount of 
activity or simply record the reading for comparison between different 
labelling procedures.
Nick



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