High Efficiency Yeast Transformation

High Efficiency Yeast Transformation

Materials

Transformation Mix (tMIX)

Reagent X1 X5 X10 X15 X20 50% PEG 4000 240 μl 1200 μl 2400 μl 3600 μl 4800 μl 1M LiAc 36 μl 180 μl 360 μl 540 μl 720 μl 10 mg/ml ssDNA 10 μl 50 μl 100 μl 150 μl 200 μl H2O 74 μl 370 μl 740 μl 1110 μl 1480 μl --- --- --- --- --- --- Final Volume 360 μl 1800 μl 3600 μl 5400 μl 7200 μl

ssDNA must be freshly denatured every time by boiling at 95°C for 10 minutes

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Method

• Start from exponentially growing cells at a concentration of 5⋅106: 10 ml for each transformation reaction;
• Prepare the required amount of tMIX
• Pellet 50 ml (=for 5 transformations) of cells 4' in a 50 ml tube at 4000 rpm at room temperature;
• Wash the cells with 25 ml of sterile water;
• Resuspend the pellet in 1 ml of sterile water and transfer to 2 ml tubes;
• Spin and resuspend in 500 μl (100 μl for each transformation);
• For each transformation, transfer 100 μl of cell suspension in a new 1.5 ml tube, ;
• Spin and discard the supernatant;
• Add 360 μl of Tmix (freshly prepared);
• Add DNA:
  • 200-500 ng for plasmids (1-4 μl of a mini prep);
  • 2-3 μg for deletion/tag cassette (5-10 μl of precipitated PCRs);
• Resuspend the cells using a vortex;
• Incubate at 42°C:
  • 5'-10' for plasmids;
  • 20-25' for multiple plasmid transformations;
  • 40' for transformations that requires a recombination event (e.g. cassettes, gap repair);
• Pellet the cells at 4000 rpm for 1' and discard the supernatant;
• If the transformation involves an autotrophy marker:
  • Wash the cells with with 1 ml of sterile water;
  • Resuspend in 200 μl and plate on selection plates;
• If the transformation involves an antibiotic resistance marker:
  • Resuspend the cells in 1 ml of YPD medium and leave for 1-2 hours at 28°C with agitation;
  • Pellet the cells;
  • Resuspend in 200 μl and plate on selection plates.