Contaminating plasmid

Sheryl White slw at med.uvm.edu
Fri Jan 14 15:10:46 EST 1994


Dr R. Dalgleish (ray at leicester.ac.uk) wrote:
: A number of us have had problems with a plasmid which seems to
: contaminate our plasmid stocks. It's ampicillin resistant and
: has a linear size of around 2.2 kb. The strangest aspect is the
: lack of sites for common restriction enzymes. It has an SstI (SacI)
: site but lacks sites for BamHI, HindIII, KpnI, PstI, EcoRI, SalI
: and EcoRV.

: It often appears as a faint band in preps of plasmid DNA from
: constructs in pUC and Bluescript type vectors.

: Can anybody suggest what it might be? I'd be very grateful for
: suggestions.

I have absolutely no idea what it is, but I've had the exact same thing
happen, TWICE.  I have only seen it with pBluescript, a plasmid of the
size you describe which doesn't cut with the appropriate enzymes.  I even
went so far as to isolate this plasmid from a low melt gel and investigate
it a bit, so here are my observations:
1)Always runs about 2 Kb and cannot be cut with enzymes which should cut
  in the MCS of pBluescript.
2)It is NOT a supercoiled form of pBluescript, as it cannot be sequenced
  using the appropriate primers (I used supercoiled pBluescript as a
  positive control, it sequenced fine).
3)Reappearance of this unusual plasmid has occurred only when I use
  Xl1-blue cells. I had reordered new plasmid from the company, it was fine
  for a while, then the mystery plasmid showed up again.  Went back to the
  new stock and grew in HB101s instead, quite some time later, there is
  still no problem.
4)I also transformed cells HB101 and XL1-Blue with the mystery plasmid. 
  The colonies formed are somewhat large, in comparison to the colonies
  formed by pBluescript.  
Anyways, good luck...I found this to be a very annoying and frustrating
episode, one which has never occurred in the use of other vectors.  I
highly recommend starting over with new plasmid and new cells, I spent way
too much time trying to figure out what the heck was going on.

----------------------- Sheryl L White  -------------------------
    Dept of Mol Physiology, Given Med, UVM, Burlington, VT 05405
       Voice: (802) 656-4433  E-Mail: slw at salus.med.uvm.edu
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