Sequencing problems

Patrick Lynch pl229 at medschl.cam.ac.uk
Mon Nov 27 09:34:47 EST 2000


Dear Phil,

I notice that you used DNA binding columns for your cleanup.  In my
experience, these bind the DNA as well as PCR artefacts that often disrupt
the sequencing.  Instead, try gel-filtration which usually gives better
results as many of the artefacts are low molecular-weight DNA molecules and
so remain in the column.  Pharmacia make pretty good ones - S-200, S-300 and
S-400 microspin columns.  The only drawback is that the sample volume is
increased but it's still concentrated enough for sequencing.  Another
suggestion is to use less PCR template in the sequencing reaction - gives
lower peaks but more readable.  I think when it comes to PCR sequencing,
less (template) is best.

Patrick

Dr. Patrick Lynch
Clinical Pharmacology Unit,
Addenbrooke's Hospital
Cambridge




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





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