What temperature does a PCR reaction boil at?
jpcd100 at mole.bio.cam.ac.uk
Fri May 18 04:40:07 EST 2001
Hi all, we have been experiencing some problems with 96 well plate
PCRs. The first observation was that occasionally some wells had less
than the 50ul reaction volume (not oil overlaid) after cycling. The
next more worrying observation was that a few had more than the 50ul
they had started with. Then most worryingly of all, when we tested our
cyclers with plates containing scattered 50ul REDTaq reactions,
surrounded by empty wells, we occasionally see that some of the
reaction has 'jumped' to a neighbouring well. Because the reaction is
red in colour, it is easy to see that the transfer has not occurred by
evaporation and recondensation.
The problem seems to be partly a sealing issue, as replacing a jumped
dummy reaction plate with one that did not previously jump in another
cycler does not result in a repeat of the previously observed jumping.
However, even if it is partly down to bad sealing, why do the reactions
boil at all? The cycler's max temp should be 94 degrees C, and even
allowing for a little overshoot it should not boil the sample. Our
cyclers have a thermistor tube option and block temperature read out,
both of which claim that are blocks are not going above 95 degrees.
Anyone got any ideas? or advice on a decent 96 well sealing sealing
John Dixon Lab 44 (1223) 333841
Dept Physiology Fax 44 (1223) 333840
United Kingdom CB2 3EG e-m: jpcd100 at mole.bio.cam.ac.uk
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