make a hot probe by PCR

eric anderson e-anderson at ski.mskcc.org
Mon Nov 25 10:16:18 EST 1996


In article <57bltj$eab at mserv1.dl.ac.uk>, andreoli at cell.biol.ethz.ch
(Christophe Andreoli) wrote:

>                         Dear  Friends,
> 
> 
>    I would like to use a 3 kb intronic fragment as a probe to screen a
> lambda library.
> Instead to waste my time in cloning this product and in labelling it by a
> nick translation procedure, I would like to make it hot during the PCR.
> Have you experienced it and how do you clean the final product in a safe
> way?

christophe,

there is no need to clone the product if you want to label it with nick
translation or random priming.  just PCR amplify the fragment of interest,
gel purify it using any method that you're comfortable with and use that
as template for your nick translation or random priming reaction.  if you
want to use PCR, the easiest way is just to add 50 uCI (usually 5uL) of
hot nucleotide (we use 32P-alpha-dCTP) to your regular PCR reaction (50uL
total).

to clean up either of these probes you can use a homemade (or commercial)
G50 column in a 1ml tuberculin syringe or use a microfuge spin column
(which is easier and is my choice) such as the Centri-Sep 10 (Princeton
Separations, Princeton, NJ, USA) or S-200 from Pharmacia.

good luck,

eric

-- 
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY  10028
(212) 639-2977
e-anderson at ski.mskcc.org



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