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Urgent PCR Questions

John Scott Meschke jmeschke at sph.unc.edu
Sat Jan 22 14:32:09 EST 2000


List-
        We recently upgraded from a very old prototype PE 4800 to a new
peltier-technology thermocycler.  In order to minimize the down time due
to the switch, I very much need to solicit protocol conversion advice.
That in mind I have come up with the following questions:
        1.  In the past we have always used thick walled 0.5ml tubes, in
the new machine these do not fit as well.  The solution suggested in the
manual is to switch to thin-walled tubes or add oil to the block in
order to tighten the fit.  The oil sounds potentially problematic and
perhaps detrimental to the machine, and I have noticed that the
thin-walled tubes tend to "craze" (stress fracture).  What is your
advice?  Do we need to switch to the thin walled tubes, is "crazing" a
problem, should we just switch to a different thick wall tube, what are
the +/- of thick vs. thin?
        2.  What are the reccommend holding times for each step of
RT-PCR?  Our current protocol calls for times of 1 hr for RT, 1.5 min
for denature, 1.5 min for annealing, and 1.5 min for extension.  I
suspect that all of these are grossly exagerrated, partly due to the
slow ramping times of our old machine and the use of thick walled
tubes.  Our new machine has ramping times of roughly 3C/sec. Our new
manual suggests that only a few seconds are needed for the denature step
and that annealing and extension will be protocol dependent.  Is there
good rule of thumb for estimating extension and annealing times?  What
holding time for denaturing provides the optimum denaturing without
affecting enzyme integrity?  How would these times differ between thin
and thick wall protocols.
        3.  The new machine has an option that allows incremental
changes in holding time over the course of however many cycles are run.
According to the manual, this is  to compensate for loss of enzyme
activity as the cycles progress. How big a problem is loss/reduction of
enzyme activity over the course of 25-40 cycles.  I assume this is
somewhat enzyme dependent.  We currently use Amplitaq or RedTaq, are
other enzymes better in this regard?
         4.  Does anyone have any other advice for converting protocols,
or improving our current protocols on our new machine?

Thank you very much in advance for any advice you can offer,


Scott Meschke
University of North Carolina





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