This protocol was submitted by Matt Kaeberlein.
Designing Gene Specific Primers:
The basic idea here is to use PCR to generate a disruption cassette that will knock-out a particular gene. To do this, you need 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 knock-out mouse, except homologous recombination is more efficient in yeast allowing for much shorter regions of homology.
As a template for the PCR reaction, you will use one of the pRS vectors (Sikorski and Heiter, Genetics 122: 19-27, 1989) as a template. If you don't have these vectors, the entire series can be purchased from the ATCC (#87538) in one kit. Maps and sequence information are available here (just scroll down the page until you get to the pRS section). You will want to be sure to use one of the integrating vectors (303, 304, 305, 306, 403, 404, 405, 406) as a template for the PCR disruption. The 3rd digit tells you which marker the vector carris 3=HIS3 4=TRP1 5=LEU2 6=URA3.
The great thing about this method is that it allows you to disrupt a gene with any of the 4 markers above using the same set of oligos. It's the template that determines what marker is present in the disruption cassette. Here's how it works: