sequenase kit

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Thu Jun 18 12:16:36 EST 1992


In article <1992Jun16.912.897 at channel1> "michael coyne" <michael.coyne at channel1.com> writes:

>She is trying to sequence ds DNA using the Sequenase kit (USB).
>Her 1.9 kb insert is contained in an M13 plasmid vector.  She is
>preparing the DNA using large scale plasmid preps (alkline lysis) and
>purifying plasmid DNA through CsCl gradients.  She has also tried
>alkline lysis mini-preps and plasmid recovery using silica.  Her problem
>is that all she gets on the sequencing gel is a large smear.
>
>Has anyone encountered a similar problem?  Any ideas as to where the
>problem might lie, or things to try to fix it?
>
>Thanks...
>
>Mike
>michael.coyne at channel1.com

Mike: I've tried many different approaches to this problem as have
many others (just look in the journal BioTechniques and you'll see
what I mean). I've had varied success trying both mini-prep and
CsCl purified DNA samples. It still appears to me that ssDNA does
work best for sequencing. Since your friend already has the clone
in an M13 plasmid (I assume this contains the f1 origin), why not
use a helper phage to produce ssDNA for the sequenase reactions?
One caution, however, is that not all clones within this kind of
phagemid will produce ssDNA. I spent quite a while trying to tweak
the smallest bit of ssDNA for sequencing from such a problem clone.


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