Difference between revisions of "Yeast Chemical Transformations"
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#*5 μl ssDNA (mix by carefully flicking the tube) | #*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. | #*5-20 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. | ||
− | # Add | + | # Add 120-150 μl PEG4000 (i.e. 2 volumes) and mix by pipetting up and down a few time |
# Immediately incubate for 15min @ 43°C. | # Immediately incubate for 15min @ 43°C. | ||
# Spin down the cells at 1600 x g for 3 min, RT and remove supernatant | # Spin down the cells at 1600 x g for 3 min, RT and remove supernatant |
Revision as of 14:51, 16 March 2022
Protocol based on Rodríguez-López et al. 2017
Chemically competent cells are available for some pombe strains in 50μl aliquots in the -80°C. If other strains are required, use the protocol Synchronized Competent Yeast Cells.
Chemical transformation
- Thaw by heating (use the 43°C bath) and then put on ice:
- Thaw Competent fission yeast cells quickly for 30 sec to RT in waterbath and put on ice
- Thaw on ice:
- HR template dsDNA e.g. produced by PCR (if frozen) or plasmid (expression vector)
- 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.
- Add 120-150 μl PEG4000 (i.e. 2 volumes) and mix by pipetting up and down a few time
- Immediately incubate for 15min @ 43°C.
- Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
- Remove supernatant and re-suspend the cells in 1 ml pipeting up and down
- Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount.
- Incubate at RT for ~16h
- Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
- 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)).
- 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.
- Thaw by heating (use the 43°C bath) and then put on ice:
- Thaw Competent fission yeast cells quickly for 30 sec to RT in waterbath and put on ice
- Thaw on ice:
- HR template repair DNA
- sgRNA plasmid (concentration should be ~200ng/μl)
- 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
- Add 145μl PEG4000 and mix by pipetting up and down a few time
- Immediately incubate for 15min @ 43°C.
- Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
- 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.
- Incubate at RT for ~16h
- Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
- Resuspend cells in 100 μl water and spread them on YES plates with 100 μg/ml Nourseothricin.
- Incubate at 32°C for at least 3 days (or even longer!)