Transform frozen competent yeast cells - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Transformation of Frozen Competent Yeast

This protocol describe yeast transformation using frozen cells Adapted from this protocol.

Material:

  • 1M Lithium Acetate (MW 65.98 g/mol): 10.2 g in 100 mL ddH2O – autoclaved
  • 50% PEG: Dissolve 50g in 30 mL ddH2O and stir, bring total volume up to 100 mL, heat and stir to combine. Filter sterilize (this might take about 10-20 min). Bubbles will disappear once you filter.
  • ssDNA: Single-stranded salmon sperm carrier DNA. We do not sonicate this DNA. It must be boiled before use, but you can refreeze 3 times after the initial boil (if you keep it on ice at all times) before you need to boil it again. Keep a few tubes in your own box for you personal use.
  • Petri dishes with solid medium for spreading transformants
  • 95-99% ethanol with flaming glass spreader

Equipment:

  • microcentrifuge
  • 42℃ water bath
  • cell spreader or glass rod
  • containers for ethanol
  • P1000 pipette
  • P200 pipette
  • P20 pipette

Method

  1. Spin down 100 µL frozen competent cells from the –80°C freezer 30 s at ~14,000 rpm or max speed of the microcentrifuge. Use one tube per transformation reaction.

  2. Remove supernatant with a pipette and add the following in order.

  • 240 µL 50% PEG
  • 36 µL 1M LiAc
  • 50 µL ssDNA
  • 34 µL DNA + water

Total volume should be 360 µL, It is usually enough to add 1 µL plasmid DNA and 33 µL of water for a normal circular plasmid prepared using a miniprep kit. If you have many similar transformations, A master mix (PEG, LiAc, ssDNA and water) can be made by leaving out your DNA. Such a mix should be vortexed lightly before being added to the cells.

  1. Resuspend the yeast cells in the mixture by vortexing well to remove any clumps.
  2. Heat shock in 42℃ water bath for 45 minutes.
  3. Spin down at 14,000 rpm or max speed for 30 s, remove supernatant with a micropipette.
  4. Add 100 µL of sterile water to the cells, resuspend by pipetting up and down with a P200 pipette and take out 10-20% of the cells to a fresh tube and save these cells in the fridge or on ice. Plate the rest of the cells on appropriate medium. Incubate plates at 30℃ for 2-4 days or until colonies appear (check after two days).
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