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)

 

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The ability to make yeast cells competent for transformation in advance allows those laboratories working with few strains to prepare large batches of cells for later transformation. This is convenient for laboratories using the two-hybrid and other similar systems. Yeast cells can be made competent for transformation by treatment with ethylene glycol and dimethyl sulfoxide (DMSO) and then frozen in small aliquots and stored at - 70^o C. While the highest transformation efficiencies are obtained with freshly grown cultures, the moderately efficient transformation of frozen competent cells saves time. This procedure has been reported to produce up to 105 transformants / µg plasmid DNA (Dohmen et al. 1991).

 

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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 1^o C freezing container (Cat. No. 5100-0001) and store at -70^o C until needed.

   TIP:

   Slow freezing results in good viability.

    
4. Add 0.1 - 5 µg of plasmid DNA and 50 µg of single stranded carrier DNA (10 mg/ml) in a maximum volume of 20 µl on top of the frozen cell suspension.
5. Place in a 37^o C 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 30^o C 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.

Preparation of frozen competent cells from the two-hybrid strain PJ69-4A using the method above routinely gave results between 0.9 - 1.0 x 10⁴ transformants/µg.