Yeast Spheroplast
Transformation Kit
Protocol
Revision No. 2210-999-6A01W

The kit is shipped at ambient temperature. However, upon receipt, it should be stored at 4°C. After the Spheroplasting Enzyme is dissolved into solution, it should be aliquoted and stored at -70°C. (See Note 1, following page).

Protocol

1. Inoculate a single colony into 50ml YPD (YEPD) Broth (catalog No. 4001-021) and grow with shaking at 30°C to an optimum cell density of 2 x 10⁷ cells/ml (OD600= 0.5-1.0). (Do not allow density to become greater than 6 x 10⁷ cells/ml as they will be difficult to spheroplast. If the cell density is greater than 2 x 10⁷, dilute to less than 6 x 106 cells/ml with YPD and incubate until cells reach the desired density. Assume that the doubling time is approximately 90 minutes).



2. In a 50 ml conical tube, spin (400 x g - 5 minutes) to pellet cells. Discard supernatant.



3. Resuspend in 20 ml sterile water; spin (400 x g - 5 minutes) to pellet cells. Discard supernatant.



4. Gently resuspend pellet in 10 ml 1M Transformation Grade Sorbitol (Transformation Grade Sorbitol powder contained in the kit should be dissolved in water to 1L and autoclaved at 15 lb for 15 minutes before using. The final concentration will be 1M). Spin (400 x g - 5 minutes) to pellet cells. Discard supernatant.



5. Gently resuspend the cells in 10 ml SCEM by rocking the tube. Gentle resuspensions and slow spins are important throughout the remaining protocol. Add 40 µl freshly thawed Spheroplasting Enzyme Mixx (see Note 1); gently mix and incubate at 30°C. Check for spheroplasting at 15 minutes. Continue to incubate until 90-95% spheroplasting is reached.




To check for spheroplasting, place 2 µl of cells in 20 µl Spheroplast Test Mix (place Spheroplast Test Mix at 55°C for 5 minutes if there is a visible precipitate before adding the cells) and observe under a microscope. Cells should be lysed and not visible or a 'ghost' membrane will be visible. Compare to 2 µl cells in 20 µl 1M Sorbitol. These should be clearly visible.




6. Spin (300 x g - 5 minutes) to pellet cells and gently discard supernatant as the cells are very fragile at this point. Because of the gentle spin, all the cells will not pellet. It is better to discard some cells on a 'light' spin than to spin 'hard' to retain all the cells.



7. Gently resuspend in 10 ml 1M Sorbitol. Spin, 300 x g - 5 minutes. Discard supernatant. Repeat this Sorbitol wash one time to thoroughly eliminate the Enzyme Mixx.



8. Gently resuspend in 2 ml CaST solution. The cells are ready for transformation.



9. In a 12 ml round bottom tube combine: 1 µl Carrier DNA, 0.1 µg-10 µg of your plasmid DNA (in a volume no greater than 10 µl), 200 µl of the yeast spheroplast cells. Gently swirl to mix. Incubate at 30°C for 20 minutes.



10. Gently add 1 ml PEG along the sides of the tube; swirl to mix. Incubate at room temperature for 10 minutes.



11. Spin, 200-300 x g for 2 minutes. Quickly discard supernatant (the pellet is not very tight at this stage). It is better to discard some of the loose cells than to achieve a tight pellet by long spins at high speeds) and gently resuspend in 150 µl SOSy and incubate at 30°C for 40 minutes without agitation.



12. Add 5 ml top agar (see Note 2, below), that is no warmer than 50°C, to the reaction tube and quickly pour on the plate agar. Incubate at 30°C 3-5 days until transformant colonies appear. The expected transformation frequency is 105-106 colonies per 1 µg of plasmid DNA. The frequency may vary due to the purity and type of plasmid used and the host cell strain.

1. Spheroplasting Enzyme Solution should be added to Spheroplasting Enzyme to form Spheroplasting Enzyme Mixx. Allow the enzyme to dissolve at room temperature for 10 minutes with intermittent agitation.




Do not heat enzyme or allow to stand at room temperature longer than 10 minutes after it is in solution. The enzyme will still be active even if it appears to be cloudy at this point. Divide into 40 µl aliquots; quick-freeze in liquid nitrogen, and store at -70°C. Thaw once before using and discard any excess.




2. The top agar should be identical to the plate agar, but should only contain 10g of agar/L. Both should contain ultra-pure 1M sorbitol (Transformation Grade Sorbitol Cat No. 2210-206 has been selected for its low toxicity and high efficiency of spheroplast regeneration) and appropriate selective factors. An agar should be selected for optimum spheroplast regeneration because different lots or grades of Agar show varying degrees of toxicity to spheroplasts. BIO 101 Agar-Y (Cat No. 4019-021) has been selected and is optimal for this purpose. BIO 101 media, selected for highest efficiency of spheroplast regeneration and growth of transformants, consists of DOBA (Drop-Out Base with Agar-Y, Cat No. 4026-022), an appropriate CSM drop-out supplement, and Transformation Grade Sorbitol (Cat No. 2210-206). For the top agar, use DOB (Drop-Out Base, Cat No. 4025-022), 10 g Agar-Y (Cat No. 4019-021) per liter, and sorbitol.