Probe labelling by PCR

Rover rover at ns1.livnet.com
Wed Nov 22 15:50:53 EST 1995


Thomas Urbig <Thomas at hastingslab.harvard.edu> wrote:

>Has onybody experience labeling gene probes by PCR and can provide 
>reaction conditions for this. It should also be possible to make single 
>stranded probes. If so, do you use a single stranded PCR product as 
>template or is a standard (double strand) PCR product as template fine. 
>Is the lower concentration of the 32P nucleotide a problem or do I have 
>to add cold nucleotide? 
>Any advice is welcome, thanks in advance
>  
>               Thomas.

	We make radioactive PCR probes on occasion. We generally don't bother
to make single stranded probes though as we haven't found a need to do
so. We perform our PCR as usual (50-100ul reaction) but add 1 uCi of
alpha dCTP 32P to the reaction (1:10 diltution of stock 10uCi/ul 32P
dCTP alpha , 3000 Ci/mmol). These probes are generally used to detect
true differentials on Northerns after using the RNA differential
display technique.

hope this helps


--Rover The Mad Molecular Biologist
rover at livnet.com
barlow at picard.evms.edu




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