Rf: PCR labelling of probes
logand at msdos.ensam.inra.fr
Wed May 24 12:01:57 EST 1995
In article <3pspbk$8m1 at homer.cs.mcgill.ca>,
Sandra Munro <smunro at cs.mcgill.ca> wrote:
>My thanks to those who responded to my query about touchdown PCR.
>Another favour - can someone provide me with a method of P32-labelling a
>DNA probe by PCR, or a reference for the method?
In a normal PCR mix I add 10ul alpha-32PdATP (approx. 3000Ci/mmol) (or
whatever nucleotide you wish) per 200ul total volume. Reduce the cold
nucleotide equivalent to one tenth of its normal concentration (ie. add to
0.02uM) and run the PCR as normal.
One of the most important points is the template DNA. For best
incorporation this must be a reamplification (as discussed on the net some
months ago). So, perform a cold PCR as normal, purify the product you wish
to use as a probe (gene-clean/ Amicon micorcon or whatever) and use a very
small amount of this (can't give a figure - depends on your intial template
amount, the amount you purify and recovery etc. etc. - so determine
empirically). I separate the labelled product from the primers, dNTPs and
salts using a Amicon Microcon 100 spin-thingy - works very well - though to
avoid contaminating your centrifuge always put parafilm around the join of
the eppendorf tube and the filter unit both for the initial spin and the
recovery spin. One final point to watch is the amount you then use - since
this method produces a high concentration of probe (note: I do not mean a
high specifc activity which is good) you have to watch the amount you use
since too much will give you a high non-specific background. I generally
get incorporation of 20-25% of the added radiolnucleotide.
David C. Logan
Logand at msdos.ensam.inra.fr
PS: No links to gene-clean suppliers or Amicon, I just think those
spin-thingies are tippity-top!
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