Silver: Integration into yeast
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Materials
- 5X TE/LiOAc
- 25 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 ~0.01 OD. For example, if you have an OD600 of 0.1, you want dilute it 100 times. (1mL into 100mL)
- 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
Day 2
- Check transformants!