Deletion problems

Hong Dang hdang at cns.neusc.bcm.tmc.edu
Wed Jun 1 20:19:33 EST 1994


In article <Pine.3.07.9406011347.A11088-b100000 at labsun1.med.uottawa.ca>, g056432 at LABSUN1.MED.UOTTAWA.CA ("jeffrey wigle ", grad stud) writes:
|> Hello 
|>  
|> 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. .
|> 
|> Cheers
|> 
|> Jeffrey Wigle
|> University of Ottawa
|> 
|> 
|> 

If your BstXI didn't cut completely, deletion would go both ways and your
primer site would be lost.  Try another clone from the same time group.

Hong



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