TA cloning nightmare

Amie Franklin amief
Tue Mar 5 23:30:05 EST 1996


Several possibilities come to mind, but the fact that you have only recovered a
truncated product suggests to me that your full length PCR product is toxic in
E. coli. Suggestions: transform and propagate your plasmid in Able C or Able K
strains from Stratagene which will reduce the  copy number of the plasmid and
the cells might not die. This worked for a friend of mine with an uncloneable
gene. As for the T/A cloning, I have used Promega's kit and it worked fine,
however they maintain that care must be taken to eliminate any potential DNAse
activity that might chew off the T end. You might want to try T addition with a
radiolabeled dTTP in order to verify all steps or to identify the bad step.

Good luck,
Amie
amief at candelab.berkeley.edu




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