Klenow question

Martin Kennedy mkennedy at chmeds.ac.nz
Tue Apr 12 21:42:54 EST 1994


In article <2od4gc$avm at server.st.usm.edu>, sywang at whale.st.usm.edu (Shiao Y. Wang) 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
> 
> 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 have a theory about many steps in molecular biology protocols.  A lot of them,
like the EDTA step in some enzyme reactions, are probably hangovers form the old
days when the enzymes were relatively impure and nucleases were rife, hence
extended reaction times were inadvisable.  That isn't such a problem with
cloned enzyme preps, and plenty of competition amongst suppliers.  The other
way superfluous steps seem to arise is when something is tried during 
development of the method, and once the method works an "aint-broke-don't-fix" 
mentality takes over.  Most of my technical breakthroughs occur when I
misread a protocol, accidentally leaving out half a dozen steps, and find that
it works just the same;-}. 
-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
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