Yeast Chemical Transformations

From PombEvolution
Jump to navigationJump to search

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!)