1. Thaw cell samples in a 37 °C water bath for 15–30 s.
  2. Centrifuge at 13,000g in a microcentrifuge for 2 min and remove the supernatant.

  3. Make up frozen competent cell (FCC) transformation mix for the planned number of transformations plus one extra, according to the protocol described below. Include an extra tube for a negative control tube for no plasmid DNA. Add this to the pellet and vortex mix vigorously to re-suspend the cell pellet. Note the difference in PEG volume from all other procedures.

    Transformation mix componentsVolume (μl)
    PEG 3350 (50% (w/v))260
    LiAc 1.0 M36
    single-stranded carrier DNA (2.0 mg ml−1)50
    Plasmid DNA plus sterile water14
    Total volume360
  4. Incubate in a 42 °C water bath for 30 min. Temperature-sensitive strains can be left on the bench overnight and then carried on to the next step.
  5. Centrifuge the tubes at 13,000g for 30 s in a microcentrifuge and remove the supernatant with a micropipettor. Transformations utilizing plasmids with prototrophic gene selection use option A and for those utilizing plasmids with eukaryotic antibiotic genes use option B.
    1. Prototrophic gene selection
      1. Pipette 1.0 ml of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break up the cell pellet and then vortex mix to thoroughly re-suspend pellet.
    2. Eukaryotic antibiotic gene selection
      1. Pipette 1.0 ml of YPAD liquid medium into the transformation tube. Vortex mix to thoroughly re-suspend pellet.
      2. Incubate for 2–3 h at 30 °C to ensure good expression from the input plasmid DNA.
  6. Plate 2, 20 or 200 μl of the cell suspension onto the appropriate SC selection medium. The 2 and 20 μl volumes should be delivered into a puddle of 100–200 μl of sterile water or YPAD depending on the selection (see Step 14). Once delivered, the inoculum is then spread with a glass rod, made sterile by being soaked in ethanol and passed through the flame of a Bunsen burner or alcohol lamp. The volume plated will depend on the efficiency of your yeast strain. Allow the liquid to be absorbed into the medium by incubation at room temperature. Cells should be plated less densely when possible, as plating density negatively affects transformation efficiency.
  7. Incubate the plates at 30 °C for 3–4 days and recover the transformants.

 

From http://www.nature.com/nprot/journal/v2/n1/full/nprot.2007.17.html

 

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