Difference between revisions of "Yeast Chemical Transformations"

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Line 8: Line 8:
 
#*[[ssDNA]] solution (salmon sperm DNA, 10μg/μl ; in top drawer freezer 7)
 
#*[[ssDNA]] solution (salmon sperm DNA, 10μg/μl ; in top drawer freezer 7)
 
#*50% [[PEG4000]] solution (in top drawer freezer 7)
 
#*50% [[PEG4000]] solution (in top drawer freezer 7)
 +
# Thaw Competent fission yeast cells quickly for 30 sec to RT in waterbath and put on ice
 
# Thaw on ice:
 
# Thaw on ice:
−
#*Competent fission yeast cells
 
 
#*HR template dsDNA e.g. produced by [[PCR]] (if frozen)
 
#*HR template dsDNA e.g. produced by [[PCR]] (if frozen)
 
# Add to the competent cells in this order:
 
# Add to the competent cells in this order:
Line 32: Line 32:
 
#*[[ssDNA]] solution (salmon sperm DNA, 10μg/μl ; in freezer 7)
 
#*[[ssDNA]] solution (salmon sperm DNA, 10μg/μl ; in freezer 7)
 
#*50% [[PEG4000]] solution  
 
#*50% [[PEG4000]] solution  
−
# Thaw Competent fission yeast cells quickly to RT in waterbath
+
# Thaw Competent fission yeast cells quickly for 30 sec to RT in waterbath and put on ice
 
# Thaw on ice:
 
# Thaw on ice:
 
#*HR template repair DNA
 
#*HR template repair DNA
Line 43: Line 43:
 
# Immediately incubate for 15min @ 43°C.
 
# Immediately incubate for 15min @ 43°C.
 
# Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
 
# Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
−
# Remove supernatant and re-suspend the cells in 1 ml  
+
# 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_-_Minimal_Mating_Medium|EMM-N]] '''or'''  
 
#*[[EMM-N_-_Minimal_Mating_Medium|EMM-N]] '''or'''  
 
#*[[EMM_-_Edinburgh_Minimal_Medium|EMM]] when working with h<sup>90</sup> or an other homothallic strain  
 
#*[[EMM_-_Edinburgh_Minimal_Medium|EMM]] when working with h<sup>90</sup> or an other homothallic strain  
−
#:Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount.
+
 
 
# Incubate at RT for ~16h
 
# Incubate at RT for ~16h
 
# 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
 
# Resuspend cells in 100 μl water and spread them on [[YES - Yeast Extract Supplemented | YES]] plates with 100 μg/ml [[Nourseothricin]].
 
# Resuspend cells in 100 μl water and spread them on [[YES - Yeast Extract Supplemented | YES]] plates with 100 μg/ml [[Nourseothricin]].
 
# Incubate at 32°C for at least 3 days (or even longer!)
 
# Incubate at 32°C for at least 3 days (or even longer!)

Revision as of 16:52, 26 November 2018

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

  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)
  4. Add to the competent cells in this order:
    • 2 μl ssDNA (mix by carefully flicking the tube)
    • 5-20 μl HR template produced by PCR. Amount depends on template concentration and also on strains used. Not-labstrain transforms much less good.
  5. Add 115 μl PEG4000 (i.e. 2 volumes) and mix by pipetting up and down a few time
  6. Immediately incubate for 15min @ 43°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
    • 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 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 43°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 waterbath 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 @ 43°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!)
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