TdT vs. Kinase
bickle at ubaclu.unibas.ch
Wed Aug 16 02:01:19 EST 1995
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
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
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