Spheroplast Transformation

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-Trp-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 SC -Trp-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

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

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. (eds.), Cold Spring Harbor Press, New York, 1999