Deletion problems

"jeffrey wigle ", grad stud g056432 at LABSUN1.MED.UOTTAWA.CA
Wed Jun 1 13:18:34 EST 1994

I'm using Qiagen purified plasmid DNA to sequence with a T7 sequencing
kit.  Generally the sequence I've got has been very clear but occasionally
I get no sequence at all from a given deletion timepoint.
The amount of DNA used for sequencing was approx. the same for the
deletions that worked and the ones that didn't -as measured by a
spectrophotometer. I denatured the plasmid with NaOH for 5 minute and
then ethanol precipitated before sequencing. The plasmid is
bluescript and the kit used for deletion was Erase-a-BASE.  I protected
with BSTXI and I opened the plasmid up for digestion with HindIII.  The
deletions looked fine when run on an agarose gel (only  one major band at
each timepoint).  i've done deletions before  and did not have this
problem but i was using single stranded sequencing(phagemid was
bluescript). I was wondering if I my problem is some inhibitor carried
over from the Qiagen prep. I would appreciate any help with this matter. .


Jeffrey Wigle
University of Ottawa

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