PCR labeling

Rover rover at livenet.net
Thu Apr 17 18:06:23 EST 1997


On 16 Apr 1997 18:25:08 GMT, mtuvin at bcm.tmc.edu (Michael J. Tuvim)
wrote:


>
>1.   Take 50 ul (500uCi, 3000 Ci/mmole, 10 uCi/ul) of alpha-P32-dCTP (Amersham
>     #PB10205 or equivalent - ain't havin' any dyes or nothin'). That
>     gives you 160 pmoles of the nucleotide. Dry it down completely in a
>     speedvac at RT overnight. Run parallel dummy: 2.5 ul of
>     2-mercaptoethanol in 50 ul of water. Check if 2-mercaptoethanol is
>     still there in the morning: sniff the dummy.
>2.   Redissolve in some (say 20 ul) water (DEPC-treated if to use with the
>     Northerns).
>3.   Add 2 ul of 5 mM solution of each cold dATP, dTTP, dGTP (gives you 

>>
>>
>
>
	I agree that this is a good  way to get an extremely high
specific activity for a probe to be used, but at quite a cost in
radioactivity, not to mention the hazard. We routinely add 50 uci of
the same isotope (alpha 32p dctp 3000 ci/mmol) . to a standard PCR
reaction (without changing any nucleotide quantities in other words) ,
this will generate a probe that is plenty radioactive for a lot of
subsequent analysis.  We have used pobes in this manner for Northerns
etc. I'm not real fond of using such a high amount of radioactivity,
if I needed a high specific activity I would probably isolate the
fragment from polyacrylamide and random label it, uses a lot less
isotope.

Rover
Eastern Virginia Medical School



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