Re-send with Address

Dr Lester Davids lester at uctgsh1.uct.ac.za
Fri Sep 10 08:46:38 EST 1999


Hi All,

I have a problem as follows. I have created a few point mutants with the
Promega GeneEditor in vitro Site-Directed Mutagenesis Kit (Cat#Q9280)
and they seem fine as they PCR fine and I've confirmed the mutations
through direct sequencing. However, when we try to express these mutants
in JM109's and try to purify them (our gene is in a HisTag vector for
purification), we get no purified protein and no activity. Recently
we've created a "safe-mutant" (ie. same aa, different last nucleotide
eg.instead of TTA we made leucine TTG) and we still have the same no-go
result. I would desperately appreciate if anyone is having the same
problem or has heard of this problem and is willing to impart advice.

Thanking y'all,
Lester Davids (lester at uctgsh1.uct.ac.za)



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