Cycle Sequencing conditions

Paul Rasmussen paul at ib3.gmu.edu
Wed Mar 10 18:06:24 EST 1999


I actually reccomend ajusting sequencing conditions depending on template
and primer.  You're right about extention times.  We use 2 minutes with a
1.2kb PCR product and are still getting usable reads in the 500-750 base
range.  Beyond that it's the resolution on our gel that gets weird, not for
lack of product.  Similar to PCR, the primers all have a specific melting
point.  If you plan on repeating the same reaction (using the same primer
in multiple samples) it can be VERY advantageous to optimize conditions
such that you are using the highest possible annealing temperature.  In
fact if you are seeing artifacts in your chromatograms, this is the first
ajustment I would try.  It could well be nonspecific primer binding.
    My understanding regarding the 60C extension temp is that since it is
so long (4 minutes) the polymerase may slowly break down at a higher temp.
We've tested 68C for 2 minutes and saw minimal differences, good or bad.
Paul

Paul Rasmussen
IB3 / George Mason University
Manassas VA
palu at ib3.gmu.edu
www.ib3.gmu.edu





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