Jaishree Muppala Chittoor
jaishre at matt.ksu.ksu.edu
Thu Dec 3 21:18:03 EST 1992
In article <1149.2b1a6f73 at nphi.fi> you write:
>Hi all bio-people in the net!
>I would like to hear opinions concerning the efficiency
>of "random priming" systems for labeling linear DNA.
>I have heard many people say that you can get a specific activity
>of 8.5 - 10 x 10E8 CPM per microgram of DNA with 32-P.
>However, I usually get about 2 - 5 x 10E7 CPM per microgram,
>regardless of DNA sequence. I use Boehringer Mannheim
>Random Priming kit.
>Here is my protocol:
>1) I denature 100 - 200 ng of linear DNA by boiling it for 3 min,
> put it quickly on ice for couple of minutes and centrifuge the
> vaporated water quickly down to the bottom of the Eppendorf tube.
>2) I use normally 50 microcurie of 32P-dATP and add cold dCTP,
> dGTP and dTTP, water, buffer and Klenow (2 U).
>3) I incubate the reaction at 37 C oven for 60 minutes, pellet the
> labeled DNA down with spermidine and single-stranded DNA and
> use it after denaturation and neutralization.
> My probes work OK, but I would like to get hotter probes. It seems
> that only about 30 - 35 % of 32P-dATP in the reaction is incorporated
> into the probe.
>Any ideas for a better protocol? Thanks in advance.
>National Public Health Institute
I use about 50-100ng of DNA and 5 ul of 5000ci/mMole 32P dCTP. I let the
reaction go overnight at 25 C. Some where I read that you get a better
probe when you let the reaction go o/n. I get very good incorporation rate.
You can prepare your own probe. It works very well. Here is the reference
Feinberg AP and Vogelstein B. (1983). A technique for radialabelling DNA
restriction endonuclease fragments to high specific activity. Anal. BIochem.
[jaishre at matt.ksu.ksu.edu]
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