reply

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.
>
>Cheers,
>
>Tapani Ronni
>National Public Health Institute
>Mannerheimintie 166
>00300 Helsinki
>FINLAND
>
> 
> 
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
for it  

Feinberg AP and Vogelstein B. (1983).  A technique for radialabelling DNA 
restriction endonuclease fragments to high specific activity.  Anal. BIochem.
132: 6-13.

Good Luck

Jaishree
[jaishre at matt.ksu.ksu.edu]



More information about the Methods mailing list