SEQUENCING PROBLEMS

[postmaster at ideaexplore.net]

Hi Phil,

As the other reply said, there are many possible reasons for the problem.
Sequencing, like any other "routine" method, is not always so routine.  The
sequence might be inherently difficult to read (polyA, or high GC), there could
be a problem with the gel composition, the sequencing reagents may be in the
wrong proportion, etc.  You can basically follow the protocol that comes with
your dye terminator kit, but you can reduce the amounts used to save money (see
http://come.to/hkeye and Biotechniques 2000 Sep;29(3):544).  Compare your
protocol with others posted online and focus on the differences to get some
clues to the problem.  Try sequencing a different PCR product at the same time
as a positive control to see if the problem is the PCR product itself or the
sequencing protocol.

Good luck,
Larry

Dept. of Ophthalmology and Visual Sciences
Chinese University of Hong Kong
http://www.ideaexplore.net