- 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.
- 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).
- inoculating several different amounts of overnight culture into different flasks allows one to choose the flask which is ready at the time of measurement.
- Centrifuge 2x 150 ml at 22C 1000-1200g for 5 minutes in flat bottom centrifuge tubes.
- Wash the cells in 20 ml of sterile water
- Wash a second time in 20 ml of 1 M sorbitol
- Resuspend in 20 ml of SCE and transfer to two 50 ml centrifuge tubes
- Add 100 μl of 2 M DTT and the optimal amount of lyticase
- See below on determining the amount of lyticase
- Incubate samples at 37C for exactly 15 minutes
- Centrifuge at 200-300g at 22C for 5 minutes
- Remove supernatant by careful aspiration
- The pellet will be soft and fluffy; not all cells will be pelleted
- Gently add 20 ml of 1 M sorbitol down the side of the tube and swirl to resuspend (do not vortex)
- Centrifuge and remove supernatant, as above
- Wash again with 20 ml of STC
- 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
- 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