Klenow question

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Tue Apr 12 11:32:18 EST 1994


In <2od4gc$avm at server.st.usm.edu> sywang at whale.st.usm.edu writes:

> 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?

Hi:

As I understand it, the EDTA stops the polymerization step by removing the 
divalent cations required by the enzyme.  Heat obviously would denature the 
enzyme.  

As far as the discussion of time, I've opted (as has most of the lab) to 
simply let the random prime reactions go 2-4 hours or until convienent to 
process the probe.  We have have no data, but the general trend is that we 
get hotter probes the longer we leave it.  In doing this we have often 
touched the elusive 1X10^9 cpm/ug.  The other thing is that we label no 
more than 75-100ng of probe with 25-50ng preferred.

Hope this helps...
David




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