[Mycology] gene deletion via PCR

Mina Falcone via mycology%40net.bio.net (by falconem from univmail.cis.mcmaster.ca)
Fri Feb 29 18:27:28 EST 2008


I am attempting to create a deletion mutant in S. cerevisiae using PCR
and inserting his3 in place of my gene of interest as the selectable
marker.  My confirmation PCR reactions (done by colony PCR) used two
sets of primers:  one set consisted of an upstream primer 200bp from
the ORF plus a downstream primer about 600bp into the selectable marker
(his3), thus giving a PCR product of approximately 800bp if the
deletion/insertion was successful.  The other set of primers are 200
and 400 bp, respectively, upstream and downstream from the original
ORF.  The wildtype gives a PCR fragment of 1200bp and the
insertion/deletion should give a fragment of approx. 2000bp.

My problem is that in 2 out of 35 colonies that I picked from the -his
selective plates the first set of primers gives the correct size of
product (800bp) indicating that the insertion is there; but, the second
set of primers gives a dominant band at 1200bp (the size of the WT gene
that I'm trying to replace) and a very faint band that is approx. the
size expected for the selectable marker.

Has anyone ever experienced this before? I am puzzled by it.  Does it
mean that there is only a partial insertion?  How can this be if the
colony originated from a single cell?  Are there revertants within the
colony?  

Should I further streak out these quasi positives or should I screen
more colonies?

Any advice would be greatly appreciated.

Mina


Mina Falcone
Research Assistant
McMaster University
Department of Biochemistry and Biomedical Sciences
Health Sciences Centre
Room 4H41
1200 Main Street West
Hamilton, ON L8N 3Z5

905-525-9140 ext 22050
falconem from mcmaster.ca




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