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Chromosomal integration

Alex Brands abbrands at artsci.wustl.edu
Fri Nov 20 16:10:06 EST 1998


On 13 Nov 1998, [iso-8859-1] Björn Johansson wrote:
> Hello,
> I have a theoretical question regarding chromosomal integration in yeast
> (S.c.).
> I would like to design a vector with PCR mediated integration ( just
> like PCR mediated gene disruption).
> This means 30-40 bp of homology on each side. Could my vector contain
> sequences that are already present
> in the yeast genome ? Would there be a large number of vectors
> integration at the wrong positions ?

You can do it, but many of the transformants will have your DNA integrated
at the wrong position.

For example, I did PCR mediated gene disruption, using HIS3 as the marker.
The PCR product had 45 bases of homology on either side.  I got ~50
transformants, but only 1 or 2 were at the right locus.  Most of them
integrated at the endogenous HIS3 locus.  If you do this, you can easily
screen the transformants by PCR, using a primer within the selection
marker, and another primer in the sequence flanking the site you are
aiming for.

Alex







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