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LiAc transformation of yeast

Creating competent yeast cells

  1. Grow yeast in 250 ml YEPD until A600 is 0.2-0.8. There are several ways to do this. For maximum efficiency, it is important that the yeast plate used to inoculate the YEPD is not too old, and that the yeast is growing well in log phase. Approach 3 below is the best approach for high efficiency. In a test transformation, yeast grown at 30°C became more competent than yeast grown at 20°C, though this should be confirmed before relying on this.
    • Inoculate the YEPD with a patch of yeast in the afternoon and grow O/N at room temperature.
    • Inoculate the YEPD with an overnight culture to A600 of ~0.1 and grow at 30°C to A600 of 0.5-0.8.
    • Inoculate the YEPD late in the day, grow O/N so the A600 reaches ~0.1. Continue growing at 30°C to A600 of 0.5-0.8.
  2. Pellet the yeast at 1800 rpm, 20°C for 3 min.
  3. Resuspend in 50ml sterile H2O at RT.
  4. Pellet the yeast at 1800 rpm, 20°C for 5 min.
  5. Resuspend in 50ml TE/LiAc at RT.
  6. Spin again.
  7. Resuspend in TE/LiAc in a total volume of 0.5 ml per 0.1 A600.

Small scale transformation in eppendorf tubes

Before starting the transformation, boil Salmon sperm DNA for 5 minutes and immediately put in an ice water bath to prevent the strands from reannealing.

  1. For each transformation mix the following. The easiest is to prepare a master mix of all the components except for the DNA, adding the yeast last so as not to shock it with a high concentration of LiAc. Prepare the mix just before adding the DNA so the Salmon sperm DNA stays single stranded.

    ComponentAmount in µl
    Competent yeast 20
    Salmon sperm DNA (2mg/ml) 20
    10x TE 11
    1M LiAc 13
    60% PEG [MW3,350] 82
    Plasmid (200 ng) in water 4
    Final Volume 150
  2. Incubate at 30°C for 30 min.
  3. Incubate at 42°C for 15 min.
  4. Add 1 ml H2O.
  5. Spin briefly in a microfuge, and resuspend in 20µl H2O to spot or 200µl H2O to plate.

Small scale transformation in multiwell plates

  1. Prepare a master mix lacking the DNA. Prepare an extra 10% to compensate for loss during the pipetting. Add the yeast last so as not to shock it with a high concentration of LiAc, and prepare the mix just before adding the DNA so the Salmon sperm DNA stays single stranded. For each well, add the following:

    ComponentAmount in µl
    Competent yeast 20
    Salmon sperm DNA (2mg/ml) 20
    10x TE 11
    1M LiAc 13
    60% PEG [MW3,350] 82
    Final Volume 150
  2. Distribute the yeast mixture into the wells of roundbottom 96-well plates, 146 µl per well.
  3. Add 4 µl of DNA (or PCR product for Gap repair) to each well, and mix well by pipetting up and down. This can be done with a multichannel pipette.
  4. Cover the plate with adhesive aluminum foil.
  5. Incubate at 30°C for 30 min in an incubator.
  6. Incubate at 42°C for 15 min, floating in a water bath.
  7. Spin the plate 5 min at 2000 rpm in a benchtop centrifuge, and remove PEG with a multichannel pipette. Hold the plate at an angle and place the tips next to the pellet to do this.
  8. Add 100 µl of H2O, then take off 80 µl (leaving less than 10 µl in the well makes the yeast hard to resuspend). Alternatively, just add 30 µl of water and proceed.
  9. Mix, and plate 5 µl on selective media.

Large scale transformation

This protocol is for 30 µg of DNA to be plated on 30 large plates, but can easily be scaled up. Before starting the transformation, boil Salmon sperm DNA for 5 minutes.

  1. In a 15ml tube, mix the following, adding the yeast last so as not to shock it with a high concentration of LiAc. Prepare the mix just before adding the DNA so the Salmon sperm DNA stays single stranded.

    ComponentAmount in µl
    Competent yeast 1600
    Salmon sperm DNA (2mg/ml) 1600
    10x TE 880
    1M LiAc 1040
    60% PEG [MW3,350] 6560
    Plasmid (30 µg) in water 320
    Final Volume 12 ml
  2. Aliquot the 12 ml into 1 ml eppendorf tubes.
  3. Incubate all eppendorf tubes at 30°C for 30 min.
  4. Incubate all eppendorf tubes at 42°C for 15 min.
  5. Spin in microfuge for 5 sec.
  6. Resuspend pellets in 750 µl H2O and pool the yeast (9 ml total volume).
  7. Add 10 µl of yeast to 10 ml H2O and plate 300 µl of this as a transformation control (efficiency = nr. of colonies X 30,000).
  8. Plate 300 µl of the final mixture onto 30 plates with appropriate selective markers.