PCR gene disruptions - methodology

Edward Braun ebraun at triton.unm.edu
Wed Nov 16 15:47:41 EST 1994


Hello all,

We are trying to perform a PCR gene disruption right now.  We have
constructed primers to the TRP1 gene that contain 15 and 17 nucleotides
of the TRP1 gene (Tm of the overlaps with TRP1 is 46.6 degrees and 45.8
degrees C) and 40 nucleotides of our gene of interest (overall Tm of
the primers are 70.4 and 67.9 degrees C).  We will amplify the TRP1
gene from YCplac22 using these primers and transform into a strain with
a trp1-deletion.

Our preliminary attempt to do this will use the following PCR rxn:

2 cycles of 1 min. 95 deg., 1 min. 42 deg., 1 min. 72 deg.
28 cycles of 1 min. 95 deg., 1 min. 65 deg., 1 min. 72 deg.

Then the PCR rxn will be used to transform the trp1-delete strain by
electroporation.

If you have been using this method to knock out genes, my question is
this: does this seem similar to the methodology you have used.  If we
get a successful gene KO I will follow this msg up with a note telling
everybody that our attempt was successful.  However, if we are using a
PCR rxn that sounds prone to falure, pls send me a msg, or post here
(It would be cool if a method for making PCR KO's that could be placed
on the net, at least in broad strokes, to help people planning this
procedure).

Thanks in advance:

:-)  Edward Braun, University of New Mexico Biology

ebraun at triton.unm.edu  



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