paranoia about cold storage and temp fluctuations?

Basavaraju Shankarappa bsh at MED.PITT.EDU
Mon May 16 09:56:44 EST 1994


> Again, Murphy was right !
> I set up more than 25 samples for PCR, and the cycler crashed.
> I have now all samples ready mixed (buffer, DNA, primers and polymerase).
> Does anyone has a recommendation, if I can still use the samples after storage
> at -20 degC ?
> I'm not shure wether the enzyme will be still OK, when stored diluted
> in the PCR-mix.

	I think biologists are plainly paranoid about storing enzymes in the 
cold all the time.  With respect to this question in particular, I have had
PCR reactions stored for about 6 hours on ice without any problems.  My 
suggestion to Sebestian is that if you are not too concerned about 
standardizing something, I would use the reactions stored at -20.  In fact,
a person in our lab had standardized PCR setups such that the enzyme+sundry
was stored in an aliquot (-20)and just add the DNA sample and go.  It had worked
reasonably well although more tests could have made the test more reliable. 
The point here is these enzymes are supposed to be thermostable and everyone
is worried about taking it off the ice or subjecting to temp fluctuations
which should not be of any consideration in concept.  I realize that in the 
old times, many enzyme preps had large quantities of contaminants that would 
have degraded the enzymes.  But I don't think it is true anymore.   
	Recently one person in our lab left a stratacooler box (hey Jim, 
here is your $250 box!) full of restriction enzymes on the benchtop overnight.
We tested the enzymes and found all of them to perform very well indeed.  
I guess this may sound like singing praises for the stratacooler at the cost
our neglect, but still the point is that people need not be
paranoid about keeping things in the cold specially PCR reagents.  
	I have to admit that people who are not familiar may take this at
face value and start not worrying about keeping things on ice including
things such as ligase.  It is difficult for many of us to take the risk and 
try to change a protocol which has been working fine.  Still I think we might
be wasting a lot of personhours worrying and implementing some of the steps
that are clearly not necessary.  I would appreciate hearing any of your 
opinions on this thread.

Raj Shankarappa
bsh at med.pitt.edu
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