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Frozen Yeast TRAFO Protocol

Frozen Yeast TRAFO Protocol



This Protocol allows You to prepare Frozen Yeast cells that are competent for transformation after thawing

After Dohmen et al. (1991) Yeast 7: 691-692. See Schiestl et al (1993)




  1. Grow cells in YPAD (10 ml per transformation) to an OD600 or 0.6 to 1.0. This represents a cell density of approximately 0.6 - 1 x 107 cell/ml.
  2. Wash the cells in 0.5 vol of 1.0 M. sorbitol, 10 mM Bicine-NaOH (pH 8.35), 3% ethylene glycol, 5% DMSO (Solution 1), and resuspend in 0.02 vol of the same solution.
  3. Freeze the 0.2 ml aliquots slowly using a Nalgene Cryo 1oC freezing container (Cat. No. 5100-0001) and store at -70oC until needed.

    TIP:

  4. Add 0.1 - 5 g of plasmid DNA and 50 g of single stranded carrier DNA (10 mg/ml) in a maximum volume of 20 l on top of the frozen cell suspension.
  5. Place in a 37oC water bath and mix every 10 - 15 sec until the solution begins to melt. Remove from water bath and mix until melting is complete.
  6. Add 1.4 ml of Solution 2 (40% PEG1000 (Roth, Karlsuhe, Germany), 0.2 M Bicine-NaOH (pH 8.35)) and mix by vortexing for 1 min.
  7. Incubate at 30oC for 1 hr.
  8. Spin down the cells at 3000 x g for 5 sec and resuspend the cell pellet in 1.0 ml of Solution 3 (0.15 M NaCl, 10 mM Bicine-NaOH (pH 8.35)).
  9. Plate an appropriate amount onto SC ommission medium into a puddle of Solution 3.



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