Silver: Integration into yeast

Back to Silver Lab

Back to Protocols

Materials

• 5X TE/LiOAc
  • 2.5 mL 1M TiOAc
  • 0.25 mL 1M Tris-HCl pH 8.0
  • 0.1 mL 0.5M EDTA
  • 2.125 mL ddH₂O

• 40% PEG / 1X TE / LiOAc
  • 1 mL 5X TE/LiOAc
  • 4 mL 50% PEG

• DMSO

• Single-stranded carrier DNA

Protocol

Day 0

• Inoculate the yeast strain into 5 ml of liquid medium and incubate overnight.

Day 1

• Measure the OD₆₀₀ and dilute it until you get to ~0.1 OD. For example, if you have an OD₆₀₀ of 5, you want dilute it 50 times.

• Grow it until you achieve an OD₆₀₀ of 0.4-0.7.

• Tranfer the cells to falcon tubes and centrifuge for 4min at 2300rpm. Decant.

• Re-suspend the cells in 10 mL ddH₂O. Centrifuge for 4min at 2300rpm. Decant.

• Re-suspend the cells in 1 mL ddH₂O into eppendorf tubes.

• Centrifuge for 10 seconds at 14000 rpm. Decant.

• Re-suspend in 0.5mL 1X TE/LiOAc buffer. Centrifuge for 10 seconds at 14000 rpm and decant. Repeat.

• Re-suspend in 50µL 1X TE/LiOAc buffer. Let sit @ 4°C until ready

• While waiting, boil ssDNA for 5min then let cool to room temperature

• Add 4µL of the digested MPs to each 50µL of cells plus 2µL ssDNA.

• Incubate for 35min at 30°C

• Add 300µL 40% PEG in 1X TE/LiOAc. Vortex well.

• Incubate for 35min at 30°C in eppendorf inverter

• Add 35µL DMSO

• Heat-shock at 42°C for 15min.

• Centrifuge the cells for 10 seconds at 14000 rpm and re-suspend in 150µL ddH₂O

• Add cells to appropriate plates according to your DNA and strain

• Put plates at 30°C

Wait a couple days before checking if you have colonies.