pGEM-T woes

Rae Nishi nishir at ohsu.edu
Fri Dec 9 13:18:26 EST 1994



When I first saw the availability of pGEM-T from Promega, I was excited
because we like to use pGEM for making riboprobes (bluescript has
background problems and the invitrogen PCR II is not a high copy number
plasmid).  The first batch seemed to work all right, but every other
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
good because a pGEM-T plasmid with a different insert will cut.  We
wasted an enormous amount of time thinking that our enzymes were bad or
that we were goofing up the digestion. Is it just my bad luck, or have
others been having problems with this vector?  Please let me know (and
please be sure to let Promega know as well).  I'm going back to PCR
II....

Rae Nishi
Dept. Cell Biology & Anatomy
Oregon Health Sciences University
Portland Oregon 97201
**that's Orygun, NOT Ora-Gone**



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