Random priming advice needed

wetsel_r at wums.wustl.edu wetsel_r at wums.wustl.edu
Tue Dec 1 06:34:55 EST 1992


In a previous article, tronni at nphi.fi wrote:
>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.

We've used anywhere from 25-250 ng.  Boil for no less than 7 mins.  Where 
we differ is the time on ice.  We go straight from the boiling water to the 
microfuge and spin it, and then it is on ice for the time it takes us to 
walk from the centrifuge (since it's non-radioactive) to our "radiation" 
area.  To state the obvious, speed is of the essence.  As soon as we get 
there, we start adding the extra reagnts.  All reagnts can tolerate the 
warmer temperature except for the Klenow and it's added last as you also 
do.

>2) I use normally 50 microcurie of 32P-dATP and add cold dCTP, 
>   dGTP and dTTP, water, buffer and Klenow (2 U).

We use 32P-dCTP but that shouldn't make any difference at this point.  
However, depending upon what we need (blot vs screening a whole library), 
we'll use anywhere from 50uCi to 200uCi for a blot and 200-500uCi for a 
library.

>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.

We use a water bath and this is where we go against published protocols.  
We let it sit there in the bath for quite a while.  We'll often set up 
probes at 1300 and not spin them over G-25 (BMB) columns until 1700, boil 
for 7-8 mins and inject it.

>   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. 

We only get 10^9 cpm/ug (equivalent) only when the radiation is very fresh. 
Once we get to about a 4-5 days past day zero, then it gets harder to get 
into the 10^9 range.

> 
>Any ideas for a better protocol? Thanks in advance.
> 
>Cheers,
> 
>Tapani Ronni
>National Public Health Institute
>Mannerheimintie 166
>00300 Helsinki
>FINLAND

I hope this helps!  Holler if you have any more questions! 

David L. Haviland
haviland at kids.wustl.edu 



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