Polynucleotide Kinase Reaction

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Wed Aug 16 06:50:32 EST 2000


In article <399A7B0E.19447B75 at ernie.orch.ruhr-uni-bochum.de>, schulffo
<Frank.Schulz at ruhr-uni-bochum.de> writes
>I want to label a trimeric oligodeoxynucleotide (CCG) on the 5'-End with
>32P via a Polynucleotide Kinase Reaction with gamma-ATP.

Nice question!

>Does anyone know, if this reacrion works well with a short
>oligonucleotide 

I know one can do hexamers and I can see no reason why a trimer
shouldn't label. One needs 20 units of T4PNK for 50pmol of an octamer
(NEB catalogue).  

>and does anyone has an idea for the separation of the
>reaction product from excess ATP and enzyme (I don't know if
>precipitation works well with short oligonucleotides).

Ethanol is no go. DE81 paper chromatography, HPLC, PAGE or maybe TLC. A
good place to look on purification is Oligonucleotide synthesis - a
practical approach, (one of the IRL Press series - old (1984) but real
bench chemistry before oligo synthesis machines!).  I doubt spin columns
will give adequate separation given that it is only a trimer.

This is the best place to ask so let's see if anyone else has done this.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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