Freezer Down - Is Taq still good?

COLINE at rulsfb.LeidenUniv.nl COLINE at rulsfb.LeidenUniv.nl
Wed Oct 11 16:36:13 EST 1995


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Date: Wed, 11 Oct 1995 10:00:50 +0100
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From: Duncan Clark <Duncan at genesys.demon.co.uk>
Subject: RE: Freezer Down - Is Taq still good?
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In article: <45e7ps$quf at mserv1.dl.ac.uk>  <COLINE at rulsfb.LeidenUniv.nl> 
writes:
> 
> Our freezer did not went down, but we were all not very carefull with
> the Taq, just took it out for half an hour on ice etc. At a certain 
moment my
> PCR (for sequencing) wouldn't work anymore so I started searching
> for new concentrations for all the ingredients except the Taq, 'cause
> that still worked in a RAPD-PCR of somebody else in the lab. It took me
> two months to come to the conclusion that the Taq was really bad, 
> and indeed with new Taq everything was back to normal; so
> the only advice I can give is try the old Taq once and if anything goes
> wrong buy new immediately. It's really sensitive.
> succes

:I can only assume you bought a v. suspect enzyme. I once took some Taq and 
:left aliqouts at RT for 6 months. I placed aliqouts back in a frezzer after 
:1 day, 2days, 4 days etc all the way to six months. They were then 
:all tested for PCR. No difference was found between brand new enzyme and 
:those that had been left out, including the six month sample.

:Duncan 
:--
:My mind's made up. Don't confuse me with the facts!


The enzym we bought is SuperTaq pproduced by HTBiotechnology LTD,
it never gave us problems and doesn't give problems anymore now we keep
it by -20C as much as possible.
I did not say the cause of our problem was HEATsensitivity.
I'm talking about openened and tens of times used taq which lost it's
activity, first it became very dependent on an exact (low) amount of Mg
(no longer the normal, wide range of possible MgCl2-conc. I was used too)
and later it completely lost activity. If anybody has an explanation for 
this I would like to know. I can only quess what happened is that
during opening (only seconds each time) it got polluted and this
reduced the activity. I think this would not have caused a reduced
activity if we would have kept it by -20C, the pollution would not
have had a chance to react. It's only quessing, but there must be a
reason why in the different labs I know everybody takes taq out -20C
only for seconds. We didn't, but now for certain we do.
I told the first story to prevent anybody else from working months
with bad taq 'cause everybody says it's not heatsensitive, if it's not
heatsensitive at least it's sensitive for something else which can 
maybe be prevented by keeping it frozen. Any suggestions?

Coline



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