Random priming advice

skspoidn at reading.ac.uk skspoidn at reading.ac.uk
Tue Dec 1 07:31:08 EST 1992


re ----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. etc
------

1) You dont say when you add your primers, although I assume 
they are added during the boiling of the dsDNA. You could try
 a slow cool from 65 rather than ice quenching for primer binding.

2) If you go from ice quench to a 37 degree reaction, you could 
again be getting a low level of random primer binding. It can 
depend on your primers, which leads me to point 3...

3) I use the Amersham Megaprime kit, or *gasp* make the reagents
 up by hand. Either way the protocol calls for the extension 
reaction to be carried out at room temperature, rather than 37.
 Also, the kit states that if using nonamer random primers, the
 extension reaction only needs 10 min. the kit (or the guy I got
 it off, I cant remember) said that if using hexamers, 
the extension reaction required 3 hours

In summary, I use 100ng DNA, boil in the presence of non/hex amers,
 slow cool from 65 to RT, add 50 uCi of a-32P dCTP etc, incubate at 
RT for 10 min/3 hours, and get realy hot probes.

Mike Poidinger
Dept of Microbiology
University of Reading

ps ever looked at the hybridization temp for a hexamer/nonamer?



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