Klenow question

Tue Apr 12 04:15:17 EST 1994

> Henry (sticknbd at ctrvax.vanderbilt.edu) wrote: 
> : When I make random-primed probes, the protocol I use 
> : says to stop the reaction after 30 to 60 minutes with 
> : EDTA (or heat), which I always do.  What would be the 
> : consequences of allowing the reaction to continue?  I 
> : cannot envision any ill effects of letting the reaction 
> : go, even if it were to go on for several hours.  Comments? 
> : sticknbd at miranda.cc.vanderbilt.edu 
> I've never understood the logic behind the EDTA step either. I skip it 
> and use the probe generated without EDTA or probe cleanup. Does anyone 
> know the logic behind the EDTA step?

I think the EDTA is included to inhibit the Klenow enzyme used in the random-
priming reaction.  The 3' to 5' exonuclease activity present in Klenow frag. 
could conceivably chew on your labeled probes and I guess that is why it is 
used for stopping the reaction, particularly if the probe is stored for 
awhile prior to use.  Heat would accomplish the same thing by denaturing the 
enzyme.  This of course would not be very critical if the probe is used right 
away in a hybridization reaction.


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