Yeast PCR Gene Disruption Using pRS Vectors

This protocol was submitted by Matt Kaeberlein.

Required Reagants:

• Disruption oligos
• PCR thermocycler and reagents
• pRS Vector template

Protocol:

1. Design gene specific primers that allow PCR amplification off of a pRS vector template.
2. Amplify disruption cassette using PCR. You can optimize your PCR conditions, but I find the following works pretty well. I use about 1/10 uL of a plasmid miniprep for the pRS vector template in a 100 uL PCR reaction. Be sure to use an integrating pRS vector (403, 404, 405, or 406) or you'll have have to gel purify the PCR product before transformation.
   • Step 1: 94C 1'
   • Step 2: 94C 1'
   • Step 3: 55C 1'
   • Step 4: 74C 2'
   • Step 5: Go to Step 2 30x
   • Step 6: 74C 6'
   • Step 7: Hold at 4C
3. Transform 30-50uL of amplified DNA into yeast using the yeast transformation protocol. If you haven't used one of the 300 or 400 series vectors, you'll have to gel purify your PCR product prior to transformation.
4. Select for the appropriate marker and verify disruptants either phenotypically or by PCR using oligos that flank your gene of interest.

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.