Factors affecting the efficiency of end-labelling by DNA kinase

[Dom Spinella]

Dominc Voon wrote:

> Hi all,
> I am going to start labelling some PCR products using DNA kinase
> (with alpha-32P dCTP) and it is quite important that I get high
> effeciency. I am just wondering if anyone could just give me a quick
> run-down on the critical factors that contribute to the effeciency of this
> experiment ? such as DNA concentration, dCTP concentration etc. Any
> pointer/comment/suggestion/advice will be much appreciated. Please email
> me directly at dvoon at cyllene.uwa.edu.au.
> 
> many thanks in advance,
> dominic

Well Dominic, I'm afraid you're in for some trouble if you try to 
end-label your DNA with T4 kinase and alpha-dCTP.  T4 kinase transfers 
the gamma phasphate from ATP to the 5' end of dephosphorylated DNA.  
If this is what you're trying to do, be sure you use gamma labelled 
ATP rather than alpha labelled dCTP.  Obtain the highest specific 
activity material you can (typically 7000 Ci/mMol) -- and use "crude" 
ATP rather than purified matrerial which is far more expensive and 
doesn't matter anyway since it is the phosphate not the nucleoside 
that you are transferring.  
Regards,  Dom Spinella