Yeast Chemical Transformations

Protocol based on Rodríguez-López et al. 2017

Chemically competent cells are available for some pombe strains in 50μl aliquots in the -70°C. If other strains are required, use the protocol Competent Yeast Cells (old version Synchronized Competent Yeast Cells has lower integration efficiency).

Chemical transformation

1. Thaw by heating (use the 43°C bath) and then put on ice:
   • ssDNA solution (salmon sperm DNA, 10μg/μl ; in top drawer freezer 7)
   • 50% PEG4000 solution (in top drawer freezer 7)
2. Thaw Competent fission yeast cells quickly for 30 sec to RT in waterbath and put on ice
3. Thaw on ice:
   • HR template dsDNA e.g. produced by PCR (if frozen) or plasmid (expression vector)
4. Add to the competent cells in this order:
   • 5 μl ssDNA (mix by carefully flicking the tube)
   • 5-20 μl HR template produced by PCR or ~1µg of plasmid. Amount depends on template concentration and also on strains used. Not-labstrain transforms much less well.
5. Add 120-150 μl PEG4000 (i.e. 2 volumes) and mix by pipetting up and down a few time
6. Immediately incubate for 15min @ 42°C.
7. Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
8. Remove supernatant and re-suspend the cells in 1 ml pipeting up and down
   • EMM-N or
   • EMM when working with h⁹⁰ or an other homothallic strain

   Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount.

9. Incubate at RT for ~16h
10. Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
11. Resuspend cells in 100 μl water and spread them on YES plates with appropriate selective agent (1 in 1000 Nourseothricin (100 μg/μl) or Hygromycin (100 μg/μl) or 1 in 500 G418 (50mg/ml)).
12. Incubate at 32°C for at least 4 days (more likely longer)

CRISPR/Cas9 transformation

This is the same protocol as above, only the added DNA and amount of PEG4000 added differs.

1. Thaw by heating (use the 42°C bath) and then put on ice:
   • ssDNA solution (salmon sperm DNA, 10μg/μl ; in freezer 7)
   • 50% PEG4000 solution
2. Thaw Competent fission yeast cells quickly for 30 sec to RT in water bath and put on ice
3. Thaw on ice:
   • HR template repair DNA
   • sgRNA plasmid (concentration should be ~200ng/μl)
4. Add to the competent cells in this order:
   • 2 μl ssDNA (mix by carefully flicking the tube)
   • 10 μl HR template
   • 10 μl sgRNA plasmid
5. Add 145μl PEG4000 and mix by pipetting up and down a few time
6. Immediately incubate for 15min @ 42°C.
7. Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
8. Remove supernatant and re-suspend the cells in 1 ml. When adding supplements (e.g. leucine, adenine etc) reduce these to 10% of the standard amount.
   • EMM-N or
   • EMM when working with h⁹⁰ or an other homothallic strain

1. Incubate at RT for ~16h
2. Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
3. Resuspend cells in 100 μl water and spread them on YES plates with 100 μg/ml Nourseothricin.
4. Incubate at 32°C for at least 3 days (or even longer!)