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Homologous recombination

Daniel Harkness harkness at bio.bu.edu
Fri Aug 18 09:14:20 EST 1995

Hello All,

The fragment of DNA that I want to use for homologous recombination is
located on a CEN/ARS yeast expression vector.  I wanted to restrict and
then gel purify this fragment away from the other vector sequences, namely
the CEN/ARS, so I can have a clean homologous recombination event.  When I 
restrict, though, the fragment and the rest of the vector are
relatively the same size, making a gel purification of just the fragment
impossible.  Further restricting the vector to make it smaller, and therefore
migrating quicker than my fragment is not possible.  Now to my real question.
If I were to do the restriction and then transform without gel purifying
my fragment away from the CEN/ARS fragment, will I run into trouble?  My 
fragment is actually a HIS reporter with specific promoter elements inserted
into the middle of the selectable marker, TRP, of this vector.  This is so
it targets to the defunct trp gene of EGY40.  I will then select for this
homologous recombination event on HIS- plates.  Please post or email me with
your suggestions/criticisms.

Very much appreciated,
Dan Harkness
Boston University
Dept. of Cell and Molecular Biology
5 Cummington St.
Boston, MA 02215
email: harkness at bio.bu.edu

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