Spheroplast Transformation
From OpenWetWare
Materials
- YPD plates
- YPD
- 1 M sorbitol 182 g/l
- 2 M sorbitol 36.4 g/100 ml
- SCE
- 1 M sorbitol 182 g/l
- 100 mM citric acid trisodium salt dihydrate 29.4 g/l
- 10 mM EDTA 20 ml of 500 mM EDTA solution /l
- final pH to 5.8 with HCl, autoclave, store at RT
- 2 M DTT
- Calf-thymus DNA (phenol chloroform purified and precipitated)
- Lyticase
- Sigma L4025
- final concentration 10,000 units/ml in
- 20 mM phosphate pH 7.5
- 50% glycerol
- store in single use aliquots at -80C
- Sorb-URA plates (per liter)
- 1 M sorbitol (182 g)
- 0.17% w/v yeast nitrogen base w/o amino acids w/o ammonium sulphate (Difco 0335-15) (1.7 g)
- 0.5% ammonium sulfate (5 g)
- 0.6 mg/l -URA dropout mix (0.6 g)
- 2% agar (20 g)
- Adjust pH to 5.8 with 1 M NaOH
- water to 900 ml
- autoclave, cool to 55C
- Add 2% w/v glucose (100 ml of a 20% solution) to final 1 liter volume
- Sorb-URA top agar (per 100 ml)
- 1 M sorbitol (18.2 g)
- 0.17% w/v yeast nitrogen base w/o amino acids w/o ammonium sulphate (Difco 0335-15) (.17 g)
- 0.5% ammonium sulfate (0.5 g)
- 0.6 mg/l -URA dropout mix (60 mg)
- 2.5% agar (2.5 g)
- Adjust pH to 5.8 with 1 M NaOH
- water to 90 ml
- autoclave, cool to 55C
- Add 2% w/v glucose (10 ml of a 20% solution) to final 100 ml volume
Material prepared just before use
- STC (per 60 ml)
- 0.98 M sorbitol 58.8 ml of a 1 M solution
- 10 mM Tris pH 7.5 600 μl of a 1 M solution
- 10 mM CaCl2 600 μl of a 1 M solution
- prepare from sterile solutions just before use
- STC + Calf thymus DNA
- Add 50 μg/ml of calf-thymus DNA (phenol chloroform purified) to STC
- prepare from sterile solutions just before use
- PEG solution (for 100 ml)
- 19.6% PEG 8000 w/v (98 ml of a 20% solution Sigma P2139)
- 10 mM Tris pH 7.5 (1 ml of a 1 M solution)
- 10 mM CaCl2 (1 ml of a 1 M solution)
- prepare from sterile solutions just before use
- SOS (for 30 ml)
- 1 M sorbitol (15 ml of a 2 M solution)
- 7 mM CaCl2 (210 μl of 1 M solution)
- 25% v/v YPD medium (7.5 ml)
- .0025% w/v uracil (75 μl of a 1% solution)
- water 7.2 ml
- prepare from sterile solutions just before use
- Microwave Sorb-ura top agar, hold at 50 C
Protocol
- 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
-
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. (eds.), Cold Spring Harbor Press, New York, 1999