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Urgent PCR Questions

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Sun Jan 23 09:54:11 EST 2000


In article <388A05B8.484B74AC at sph.unc.edu>, John Scott Meschke
<jmeschke at sph.unc.edu> writes
>        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?

All 0.2ml microtubes for PCR are thin wall as are plates. I would use
0.5ml thin wall from a good manufacturer of such tubes. They really
shouldn't craze during ordinary PCR.

>        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.

Thick wall tubes require longer hold times simply because the
temperature in these tubes lags a long way behind the temperature of the
block. For glass capillary based machines people use 0sec hold times at
annealing and denaturation. For thin wall tubes in a modern machine,
PE9700 and PE2400, we use 15secs and have on occasions reduced that to 5
secs without problems. 

>        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?

It depends to some degree on your denaturation temp. If you use 95C and
30-60secs at each cycle you will get close to the half life of the
enzyme. At 15 secs at 94C you should not see this problem. We do however
as a matter of course autoincrement enzyme mixes i.e. Taq + a proof-
reading enzyme for both Long PCR and RT-PCR. On a 9700 something like 5
secs per cycle at 68C but only after say the first 15-20 cycles have
been dome normally.

>         4.  Does anyone have any other advice for converting protocols,
>or improving our current protocols on our new machine?

If you stick to the annealing temperatures defined for your oligos and
you aren't working with really tricky PCRs that only work once in a blue
moon for no apparent reason (or you aren't doing the appropriate
dances/balck magic as discussed a few weeks ago in this newsgroup) then
you really shouldn't have any problems.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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