Contaminating plasmid

Dave Harry deh at s27w007.pswfs.gov
Sat Jan 15 13:38:14 EST 1994


>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
                                          ^^^^^^^^^^^
Ditto my experience.

>size you describe which doesn't cut with the appropriate enzymes.  I even
                         ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Again, ditto.
  
>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
   ^^^^^^^^^^^^^^
Again, ditto.

>  new stock and grew in HB101s instead, quite some time later, there is
>  still no problem.

Nearly ditto.  I used DH5-alpha.

I shared Sheryl's frustration in her experience.  This "mystery" band
caused endless grief a couple of years ago.  It kept cropping it's ugly
head and caused mass confusion.  I also made some hand-waving arguments
about leaky f1 origins (as in earlier posts) and further speculated that
it might have something to do with XL1-Blue's tet-containing episome.  In
desparation, I called Stratagene for advice, only to learn they'd never
seen this (so therefore we must be some foolish incompetents?!) and could
offer no insights.

In short, I never did learn what it was.  *BUT*, the problem did indeed
disappear after transforming my Bluescript vectors into different a
different host (DH5-alpha).  I now use XL1-Blue only when absolutely
necessary to carry out certain procedures, and then get my plasmids the
H--- out of there as fast as possible and into another host.

Until I saw this discussion, I thought we were the only ones who'd
encountered this problem.  Thanks to all for sharing!!

David Harry                       Institute of Forest Genetics
deh at s27w007.pswfs.gov             USDA Forest Service, Pacific SW Station
Phone:  510/559-6439              PO Box 245
FAX:    510/559-6499              Berkeley, CA 94701

 



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