96 well sealing problem.

Chris Bacot bacot at bio.fsu.edu
Thu Nov 16 16:29:22 EST 2000


Guy, 

Try MicroAmp clear adhesive films from Perkin-Elmer ($1/ PCR plate), 
Fisher and Phenix plastics also have a similar product, but I find the P-E
product is 
more robust with respect to sealing integrity around edges and at high
temps for 
prolonged periods of time. Hopefully this will correct the problem, if not
read 
on..... 

Given that this event occurs regarless of sealing material, the cause might
be 
attributed to your plate or perhaps the thermocycler (or a synergy of the
two): 

Inherent problem with plate theory:  Something about the plate (moisture,
grease, 
etc.) is introduced to the plate at set up time only near the periphery.
Poor seal at 
the edge and therefore samples in these areas evaporate during
thermocycling.  Bad 
lot of PCR plates from vendor, i.e. doesn't allow sealant to stick near
edges.  Try 
different plates. 

Inherent problem with thermocycler:  There is a distinct temperature gradient 
between periphral wells and internal wells of thermocycler.   The sealing
strip 
(regardless of kind)  therfore heats and cools in a non-uniform fashion
causing 
"vents" to occur around periphery resulting in evaporation of samples at
edges of 
plate.   See if the plates and sealants that are problematic on your
thermocycler are also problematic on other thermoclycers.  If they work
fine on others, then it is likely you need to replace your block or peltier.

If you are in a crunch to move samples through and this is your sole
thermocycler, 
use only internal wells on you PCR plate till you can invesitgate further. 

Best regards, 

Chris 
 

***********************************************
Christopher M. Bacot

DNA Sequencing Facility			  
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Department of Biological Science		  
Florida State University			  
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