Cyclers for automated sequencing.

Brian Foley brianf at med.uvm.edu
Wed May 25 10:01:17 EST 1994


Dear Researchers:
	We have purchased an ABI 373 automated sequencing apparatus.  We
currently use a Perkin-Elmer 9600 thermal cycler to perform the cycle
sequencing reactions needed to produce the labeled, terminated DNA 
ladder.  Our PE 9600 is also used for other methods and is often tied up 
and not available for sequencing.  Thus we need to buy a second cycler
and are considering a capillary-tube cycler as an alternative.
	The PE 9600 works very well, but 25 cycles take just under 2 
hours.  We have a capillary tube cycler that can perform the same 25 
cycles in less than 1/2 hour.  However, ABI is quite specific in 
recommending ramp rates and we are wondering if this will affect the
sequencing reactions.

	Because we have both machines available here, I plan to test them 
both empirically to see if they give equivalent results.  However, if any 
of you have already doen this you could give me some advice:

	1) Is it necessary to use a different thermal profile in the 
capillary tube cycler than the one used in the PE 9600?

	2) Can I add BSA to my sequencing reactions to prevent the DNA 
from adhering to the glass capillary tubes?  If so do I need a protease 
treatment prior to loading the sequencing gel?

	3) Is it possible to cut the reaction volume to 10 microliters or 
to use two 10 microliter capillary tubes per reaction?  Or do I need to 
use the larger capillary tubes?

--
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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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