Q: Can you random prime a 250bp PCR product?

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Jul 31 04:59:37 EST 1998


Hi Makoto,
While there is no problem in ramdom priming of such a fragment, I wonder why
you want to do RANDOM priming at all. As you have a PCR probe, you have two
specific primers in your hands that are great for labelling the DNA. Do it as
for random priming, but use one of your primers instead of a hexanucleotide
mixture. That is: denature, anneal, extend. You can use your PCR conditions,
but you will possibly get higher specific activity with:

boil DNA for 5min
quick-chill by placing in ice-water for 10min
add primer of choice. exact amount is uncritical as long as it is in excess
add radioactive dATP (or the nucleotide of choice), and add dNTP mix without dATP
add klenow enzyme (1unit)
let stand at room temperature for 1h

You´ll get one labelled strand only, of course. Use both primers for labelling
both strands (and increasing specific activity)

hope this helps,
Frank



Makoto Kamei wrote:
> 
> Hi all,
> 
> I am about to screen a mouse genomic DNA library constructed in EMBL3
> lambda vector. I have ~250bp PCR product made from mouse genomic DNA
> template. Is it possible to use this product as a probe for the screening?
> Is it possible to random prime this product to make a probe? Any help
> appreciated. Thanks a lot in advance.
> 
> Cheers,
> 
> Makoto Kamei
> Makoto.Kamei at anu.edu.au



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