Team:Aachen/Wetlab/Protocol18

Transformation of S. cerevisiae:

Material:


  • 1 M lithium acetate (LiOAc) → autoclave for 15 min.

  • 0.1 M LiOAc

  • 50 % PEG 3350 in water → autoclave for 15 min.

  • single stranded carrier DNA → fish sperm DNA, prepare 2 mg/mL solution in dH2O and denature for 5 min at 95 ℃.


Procedure:


  1. preparation of DNA-solution: Combine 0.5-1 ug each of desired fragments (ratio of 1:2 for fragments containing marker elements to fragments without marker element), bring to a total volume of 34 uL with dH20 (for plasmids, use 100-300 ng).

  2. prepare transformation-mixture (can be scaled up and prepared as master mix):

    • 240 uL 50% PEG 3350.

    • 36 uL 1M LiOAc.

    • 50 uL carrier DNA.

  3. centrifuge aliquot of competent cells (16,000xg, 15 s) → e.g. cryos of competent cells.

  4. resuspend cells in 34 uL (plasmid-)DNA- solution.

  5. add transformation-mixture and vortex gently.

  6. incubate at 30 ℃ for 30 min.

  7. invert tube, then incubate for precisely 30 min at 42 ℃.

  8. centrifuge cells (16,000xg, 15 s).

  9. A) if antibiotic marker is used: resuspend cells in 500 uL YPD and incubate at 30°C and 200 rpm for 3-4 h, afterwards spread on selection medium
  10. B)if there is no antibiotic marker: resuspend pellet in 100 uL A. bidest, spread on selection medium.