Cycle sequencing large inserts

Sasha Kraev bckraev at
Thu Nov 24 10:31:24 EST 1994

14 kb is by far not the limit of cycle sequencing. However, it seems that
supercoiled DNA is used less efficiently, as opposed to the linear of the
same molar amount. We routinely sequence 120 ng of lambda DNA, using however
32P or 33P labelled primers. Second, it is quite important, that your annealing
temperature for an insert-specific primer is optimal. Longer primers, 
that allow a two-step cycling, tend to perform better. So, 
 1) Try cutting your plasmid before sequencing , 
 2) Try using labelled 24-26 bp primers. 
With these changes you should be able to sequence from about 200 ng of your DNA.
Good luck, Sasha

More information about the Methods mailing list