IO00865@MAINE.BITNET (01/16/91)
I have been trying to use Stratagene's pBluescript II SK (+-) vector for some subcloning. So far I have only sequenced one clone, but part of the polylinker region and part of the insert appear to have been scrambled. Has anyone else seen anything like that with this vector? Is it worth sequencing more clones in search of a good one? Any comments would be appreciated. Thanks, Ethan Strauss Io00865@maine.bitnet
DUSAN@physik.uni-bielefeld.dbp.de (01/18/91)
IO00865@MAINE says: >I have been trying to use Stratagene's pBluescript II SK (+-) vector for some >subcloning. So far I have only sequenced one clone, but part of the polylinker >region and part of the insert appear to have been scrambled. Has anyone else >seen anything like that with this vector? Is it worth sequencing more clones >in search of a good one? Any comments would be appreciated. > > Thanks, > Ethan Strauss > Io00865@maine.bitnet I have got similar results using the Klenow fragment after ExoIII/ExoVII treatment of an insert (in order to introduce nesting deletions). However, this was only true for one sequenced clone out of four or five, I think. The deletions (I think I missed about 10 bases) are probably due to the still abundant but low exonuclease activity of the Klenow fragment. regards, Dusan Zivadinovic Department of Genetics University of Bielefeld W 4800 Bielefeld Germany email: dusan@physik.uni-bielefeld.dbp.de