This protocol was submitted by Matt Kaeberlein.
Required Reagants:
Protocol:
Designing Gene Specific Primers:
The basic idea here is to incorporate ~40 nucleotides of homology to the yeast genome into your oligos. This homology will determine where the marker gene integrates into the genome - hopefully taking out the sequence for the gene you want to disrupt. This is the same principle used in making a knockout mouse, except homologous recombination is more efficient in yeast allowing for much shorter regions of homology.
The other principle we will use is to
Notes:
The efficiency of this method for generating gene disruptions is quite variable. The most important factor seems to be the allele of the marker present in your starting strain. If the marker you are using is present as a point mutation, gene conversion will occur frequently and a high fraction of marker positive transformants will not carry the gene disruption. You can increase your efficiency by using another marker or by using a Kan disruption cassette. In W303, using 40 nts of homology in the disruption oligo, I usually get from 5-30% disruption efficiency.