pGEM-T woes

Timo Hiltunen bltihi at uta.fi
Tue Dec 13 02:02:32 EST 1994


Rae Nishi (nishir at ohsu.edu) wrote:
..
: batch we've gotten has had problems.  For example, one lot had an
: unacceptably high background of white colonies when the plasmid alone
: was used to transform.  Another lot seemed to be missing the T-
: overhang so that we couldn't get even the control insert to work.  More
: recently, we've had a more insidious problem... two clones (with
: different inserts, each prep done by a different person in the lab)
: that we isolated are missing the T7 end of the vector; that is, none of
: the enzymes that are in the multiple cloning site on the T7 end of
: where the insert should go will cut.  We (now) know all our enzymes are
..

I've used pGEM-T with generally acceptable results, but I use a 
ligation time of about 24 hr.
However, I noticed in some of the minipreps that the isolated plasmids
had major deletions somewhere near their MCSs. This concerned only the 
white colonies and never the faintly blue ones which were usually correct
ones (in some cases, the white ones also were correct). 
The proportion of deletion-containing plasmids seems to vary between 
lots, and right now I have a good kit in my hands (not literally).

Anyhow, Promega's products are much more reliable than Clontech's:
I used the PCR MIMIC Construction Kit, and it had a significant deletion
in the template DNA. After complaining, they sent me another tube.....
it had the same deletion. 

TH



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