Spheroplast Transformation

Spheroplast Transformation

1. Pick a single colony from a YPD plate containing the correct strain into 5 ml of YPD medium and grow overnight at 30C with shaking. Do not inoculate from a plate stored at 4C.
2. Add 200-800 μl of the overnight culture to 350 ml of YPD medium in a 1 liter flask and grow 14-18 hours to a final concentration of 5 x 10⁷ cells/ml (mid log phase).
   1. inoculating several different amounts of overnight culture into different flasks allows one to choose the flask which is ready at the time of measurement.
3. Centrifuge 2x 150 ml at 22C 1000-1200g for 5 minutes in flat bottom centrifuge tubes.
4. Wash the cells in 20 ml of sterile water
5. Wash a second time in 20 ml of 1 M sorbitol
6. Resuspend in 20 ml of SCE and transfer to two 50 ml centrifuge tubes
7. Add 100 μl of 2 M DTT and the optimal amount of lyticase
   1. See below on determining the amount of lyticase
8. Incubate samples at 37C for exactly 15 minutes
9. Centrifuge at 200-300g at 22C for 5 minutes
10. Remove supernatant by careful aspiration
    1. The pellet will be soft and fluffy; not all cells will be pelleted
11. Gently add 20 ml of 1 M sorbitol down the side of the tube and swirl to resuspend (do not vortex)
12. Centrifuge and remove supernatant, as above
13. Wash again with 20 ml of STC
14. Resuspend in 6 ml of STC + 50 μg/ml of calf-thymus DNA and combine into a single tube

YPD plates YPD 1 M sorbitol SCE 2 M DTT Lyticase STC STC + Calf thymus DNA PEG solution SOS Sorb-URA plates

1. Yeast Artificial Chromosomes, Green E.D., Hieter, P., and Spencer F. A., chapter 5 in Genome Analysis: A Laboratory Manual, Vol. 3, Cloning Systems, Birren et al. (ed.), Cold Spring Harbor Press, New York, 1999