TdT vs. Kinase

[Tom Bickle]

In article <40q8oa$cka at synapse.bms.com>, Steven Goldberg
<goldberg at bms.com> wrote:

> Until recently our lab has been using T4 kinase + gamma 32P-ATP to end- 
> label our oligonucleotides.  However, we tried terminal deoxynucleotide 
> transferase and found our probes were labeled to a much higher specific 
> activity. Another advantage is that you can use a single type of 
> radionucleotide (alpha 32P-dATP) for all other types of labeling you 
> might have to do, including random-priming.  Has anyone had the same 
> experience that we had as far as getting hotter probes--also, are there 
> any known disadvantages to using probes prepared using TdT vs.kinase?
> 
> Thanks for your responses
> 
> Steve

The main reason your probes are hotter is that you are putting poly (dA)
tails on your oligos- multiple hot phosphates. This might cause
hybridization problems in some cases. You can get a single incorporation
by using an alpha 32P-ddATP or by using alpha labelled rNTP and alkali
hydrolysing back to the single adduct. But then T4 kinase would probably
be less trouble.

-- 
Tom Bickle, Department of Microbiology, Biozentrum, Basel University
Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
+4161 267 2120, Fax: +4161 267 2118  bickle at ubaclu.unibas.ch