Yeast Spheroplast Transformation Kit



A somewhat more involved procedure than the Alkali-Cation transformation protocol, but results in highest frequencies of transformationÉas many as 10% of spheroplasts made from high efficiency strains can be transformed with minimal nuclear fusion. This is essential for procedures requiring the greatest number of transformants, such as screening cDNA expression libraries for complementation, or for construction of YAC libraries. Each preparation produces sufficient spheroplasts to do 5-20 transformations with up to 5 µg of DNA; and each transformation can produce up to 105 to 107 transformants per DNA sample depending upon the strain.

The protocol in brief:

  1. Wash log phase yeast cells in sorbitol and resuspend in SCEM.
  2. Make 90% spheroplasts with Spheroplasting Enzyme Mixx.
  3. Wash spheroplasts in sorbitol and resuspend in CaST solution.
  4. Transform spheroplasts with carrier and plasmid or YAC DNA.
  5. Gently wash and plate spheroplasts in regeneration top agar with appropriate selection media.



Cat No.DescriptionSize/Vol.
2210-200Yeast Spheroplast Transformation Kit250 trans
2210-201SCEM, sterile solution275 ml
2210-203CaST, sterile solution55 ml
2210-205Carrier DNA, 10 mg/ml, sterile250 µl
2210-202Spheroplast Enzyme25,000 units
2210-210Spheroplast Enzyme Solution1.1 ml
2210-207PEG, sterile solution250 ml
2210-204SOS, sterile solution40 ml
2210-208Spheroplast Test Mix1.25 ml
2210-209Control Yeast Strain1 slant
2210-211Control Plasmid DNA300 µl
2210-206Sorbitol, Transformation Grade
(powder to make 1 L of 1M solution)*		185 g
Shipping and Storage: Ambient
*Transformation grade Sorbitol powder contained in the kit should be dissolved in water to 1L and autoclaved at 15lb for 15 minutes before using. The final concentration will be 1M.



Protocols




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