Quick and Dirty Yeast Transformation

This protocol was adapted from “‘Quick and Dirty’ Plasmid Transformation of Yeast Colonies,” Techniques and Protocols 2, in Methods in Yeast Genetics, 2005 edition, by David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

MATERIALS

  • β-mercaptoethanol, >98% (~17 M); Sigma
  • salmon sperm DNA, 5 mg/ml
  • Lithium acetate, 2 M (less than 1 month old, preferably fresh)
  • Plasmid DNA
  • Polyethylene glycol (PEG) 3350 (Sigma), 50% (w/v) sterile

PROTOCOL

  1. Start denaturing Salmon sperm: 5 min boil, then put on ice.
  2. Prepare the following transformation master mix (good for 10 transformations)
    • 200 μl 2 M lithium acetate
    • 800 μl 50% PEG-3350
    • 7.7 μl β-mercaptoethanol
  3. For each transformation, aliquot 100 μl of master mix into a fresh microcentrifuge tube. Add 6 μl of 5 mg/ml denatured salmon sperm DNA and 1 μg of plasmid DNA.
  4. Suspend one large yeast colony (from a fresh streak) in each transformation reaction and vortex. (Alternatively, ~250 uL { >5e7 cells} of saturated culture can be spun down and used)1
  5. Incubate for 30 minutes on a rotator at 37°C.
  6. Centrifuge cells for 5 minutes at 3000 rpm, discard the supernatant in

TROUBLESHOOTING

Problem: Inefficient transformation: use fresh colony

1.Saturated cultures can be used, but growing cultures work much better.

NOTES

Using a 1ml pipet tip works well to pick up cells