pcDNA 1.1 Amp rearrangements
Harry.Witchel at Bristol.ac.Uk
Sun Sep 21 13:34:19 EST 1997
Good day Popes of Molecular Biology --
I have been trying to clone various genes around the lab into
InVitrogen's pcDNA 1.1 Amp, and I regularly find that a fair number of
colonies look like they have my insert (upon digestion), but that the
vector is the wrong size. The vector should be 4.8 kb when linearised
(and in my control lane the linearised vector runs at 4.8 kb), but in
these strange transformants the cut out vector invariably has a
molecular weight of ~3.5 kb. I believe that band is the vector because
there are no bigger bands (eg anything at 4.8 kb), and the other bands
will correspond to what I would expect for my insert. I do not think my
enzymes are cutting the vector anywhere besides the multiple cloning
region (the enzymes are listed by the manufacturer as unique cutters).
The irritating thing is that in my hands pcDNA 1.1 Amp behaves as a
medium copy # plasmid, whereas this putative deletion product acts as a
high copy # plasmid.
Has anyone seen this before or know what it is?
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