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ura3 inactivation

Sandra Vajic sunny at eunet.yu
Mon Sep 8 04:41:12 EST 2003


Dear all,

I am trying to inactivate P. pastoris URA3 gene using 5-FOA for a
selection.  I tried to disrupt the gene by introducing ura3 PCR
fragment of 430bp with two stop codons- one at 5' and one at 3'
end. It was unsuccessful.  Then I cloned the same PCR fragment of ura3
into pUC19 and cut it with NcoI to have inactivation cassette with
180bp of P. pastoris ura3 gene at 5' end, 280bp at 3' end and in the
middle entire pUC19 sequence. For the transformation I chose
electroporation with different amounts of DNA (0.1, 0.5, 1, 5ug). It
was also unsuccessful.  I also tried to obtain spontaneous mutants as
I read in a few publications but all of them grew on minimal plate
with 1g/L 5-FOA but also on minimal plate without uracil.

I would appreciate if somebody has a solution or suggestion for my
problem. I would be also thankful if somebody have the whole
P. pastoris ura3 gene and is ready and able to share it.  I am
Ph.D. student from Belgrade and this experiment is important for my
Master thesis.  Thank you in advance

Lidija

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