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

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.

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

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