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PCR KO strategy

John R. Geiser geiser at JHUNIX.HCF.JHU.EDU
Fri Nov 4 19:36:58 EST 1994


Just thought I would weigh in with my two cents.  I've done the PCR KO once
with a small variation.  I did not use a delete of URA3, only ura3-52.  I
screened through 30 URA+ positives by PCR and found 4 that integrated at my
gene.  All 4 gave me the phenotype I expected after I had identified them
with PCR.

The variation I used is shown below:

OLD technique

PCR up marker (URA3) with homology to region you want to delete.  45 bases
of 5' of gene and 45 bases of 3' of gene.  Gives you the PCR product shown
below.

5' YFG - URA3 - 3' YFG    YFG = your favorite gene

Transform and select for URA3 or other marker you are using.


NEW variation

My improvement has been to change the part that is PCRed.  Oligos are made
same way, but template is different.

5' YFG - cyc1-terminator - URA3 - cyc1-terminator - 3' YFG

Transform, select as usual.  5FOA to regain URA3 marker.  The nice part
about it is that you create a strain that allows you to keep the marker if
you want (for further genetic experiments) or lose the marker and regain
its use (for libraries, etc).  Also takes advantage of 5FOA and does not
require plasmid construction of the complete knockouts.

Rate of recombination can be kept low by the amount of the direct overlap
in the cyc1-terminator.  No reason why cyc1-terminator has to be used.
That was just the construct I had available to try this out.
cyc1-terminator can be exchanged for GAL1 to drop GAL1 promoter in front of
any gene.  ETC, ETC, ETC.

I've heard anectdotal evidence that HIS3 works better than URA3, but have
had no experience myself.  It also takes away ability to rid yourself of
the marker.  ie no 5FOA

Hope you find this useful or at least entertaining!

Anyone else have any other variations on this theme??

JOHN

______________________________________________________________________
John R. Geiser                         geiser at jhunix.hcf.jhu.edu
Department of Biology, Mudd Hall       Phone:  (410) 516-7293
Johns Hopkins University               FAX:    (410) 516-5213
Baltimore, Maryland  21218
USA                                    Fix the Problem, not the Blame!





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