microtiter plate PCR?

steinger at urz.unibas.ch steinger at urz.unibas.ch
Wed May 12 14:13:52 EST 1993

 I am running RAPD/PCR (with single 10-mer primers) in microplates and can
share some of my experiences with you.
First, I would like to warn you of the use of hot air ovens
for PCR. My own measurements with two thermocouples revealed
that the temperature within the oven can greatly vary according
to position. In a test for reproducibility of the RAPD amplification
I observed significant positional effects between opposite edges
of a single microplate. PCR with longer primers might be less
sensitive to temp. but you should check this first.
There are several block thermocyclers suitable for microplates
available on the market (e.g. MJ Research, Techne, Hybaid, PE with
expensive disposables). In my lab I use the MJ Research PTC-100-96V
machine with the new block that accomodates V-bottom polycarbonate 
plates (U.S. Scientific Plastics). Polycarbonate plates are not being
deformed at high temperatures and therefore ensure proper contact
and thermal transfer between block and sample wells. Sample volume
can be as low as 10 ul in V-bottom plates. In the MJ Res. machine
I measured a temp. gradient between center and corner of 0.3 degrees
at annealing temp. (36 degrees) and 1.5 degrees at denaturing (92 deg)
No positional effects were detected in the RAPD banding profile.

I hope this information will help you. Good luck!

Thomas Steinger
Botanical Institute
University of Basel, Switzerland

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