GMS-cometition/probe quantitation

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Feb 21 04:12:02 EST 2000


Moez Torki wrote:

> Hello biomolers,
>
> I'm performing GMS using a 160 pb DNA probe. The probe was PCR amplified,
> EcoRI restricted, G-50 purified, 32P end-filled and agarose gel purified.
> Doing that way I've got probes with very *hot* : 7 million cpm. I used
> 30,000 cpm for the GMS and had nice shift. Now I'd like to perform
> GMS-competition with unlabelled fragment, but I don't know exactely how
> many pmoles of my labelled probe I'm actually using ... Hence I'd like to
> know if there is a formula to convert the cpm (of a clean probe) to pmoles
> of DNA ?
>
> Thanks for any hint,
>
> Moez
>
> ---

No, unless you exactely know the amount of DNA you get after purification.
Using G-50 to remove unincorporated nucleotides, you´ll get a good recovery,
between 90 and 95% usually. The critical step is gel purification, where the
loss of DNA can be very high. Depending on the method you can recover between
30% and 80%. Thus, there´s no easy formula to apply to convert cpm to pmoles.

What I would do to quantitate DNA is the following:
1. After labelling (but before any purification), take an aliquot of the
sample and precipitate with TCA.
2. Count in scintillation counter
3. Knowing the size of the aliquot and the amount of input DNA, calculate
specific activity of the probe in cpm/ng
(Example: your aliquot was 1ul of 100ul, and you had 1ug of DNA for labelling
=> your aliquot will be 10ng.;
Scintillation counting says you have 200.000cpm in it => specific activity
will be 20.000cpm/ng)
specific activity will not change during purification, of course.
4. use this value to convert your amount of assay DNA to ng. (Example: you
use 30.000cpm for the assay, with specific activity of 20.000cpm/ul => your
input DNA will be 1.5ng
5. Use this result to calculate the amount of competitor DNA you need. There
is no need to calculate it in pmoles, as the unlabelled fragment is of the
same size (and thus MW).

Frank







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