Thermal cycler sequecing...

Leslie A. Johnston-Dow ldow at
Thu Apr 2 10:57:43 EST 1992

(Seems to me my day for throwing in my .02)
Have you tried nesting the sequencing primer? In my hands this
gives a much better sequencing reaction. If you nest the primer
you remove any 'primer dimer' from competition for your sequencing
primers. In my hands, this has taken care of much of the noise
that you see in the first 50-100 bases of the sequencing reaction.
Now I must make the caveat that I am using a Taq cycle sequencing
protocol which greatly enchances the difficulty from this problem.
But I have also seen nesting help in non-cycling sequencing reactions.
(In addition, I am using our fluorescent sequencing system, but a 
similar principle applies) 
Hope this helps!

         Leslie A. Johnston-Dow, Ph.D.
Diagnostic R&D               Applied Biosystems, Inc
          ldow at

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