Quantitating Radioactive label in DNA and RNA

Stephen R. Lasky Stephen_Lasky at brown.edu
Wed Jun 29 17:34:55 EST 1994


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
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America may be unique in being a country which has leapt from barbarism to
decadence without touching civilization:  John O'Hara
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