SEQUENCING PROBLEMS

Philip O'Brien obrien at central.murdoch.edu.au
Mon Nov 20 22:01:32 EST 2000


We have been using dye terminator cycle sequencing and getting poor
results.  The sequence contains a lot of N's.

The template is a PCR amplicon (shows only a single band on agarose)
purified using a Wizard cleanup column (Promega).  Sometimes we use
glassmilk instead.

The sequencing reaction uses approx 200ng template.  When we measured
concentration by fluoresence (Hoefer dye)  the concentration was lower than
expected.  From electrophoresis band intensity we know it contains at least
200ng.

The sequencing reactions were cleaned up by ethanol precipitation.  We have
been really careful with the precipitation protocol to ensure that the
unincorporated dye terminators do not precipitate out.

However the final result shows too many mistakes, too many N's in the
sequence.

Is there anything obvious that I'm missing, or has anybody else had similar
problems?

phil

Philip O'Brien
Division of Science
School of Biological Sciences & Biotechnology ,
Murdoch University,
Murdoch WA 6150,
AUSTRALIA

Ph Nat     61-8-9360 2785
Fax         61-8-9310 7084
http://www.murdoch.edu.au/home.html

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