Spheroplast Transformation

Materials

• YPD plates

• YPD

• 1 M sorbitol 182 g/l (Sigma S7547)

• 2 M sorbitol 36.4 g/100 ml

• SCE (per liter)

  • 1 M sorbitol (182 g)

  • 100 mM citric acid trisodium salt dihydrate (29.4 g)

  • 10 mM EDTA 20 ml of 500 mM EDTA solution

  • final pH to 5.8 with HCl, autoclave, store at RT

• 1 M DTT solution Sigma 43816

• Calf-thymus DNA (phenol chloroform purified and precipitated)

• Lyticase

  • 20 mM phosphate pH 7.5

  • 50% glycerol

  • store in single use aliquots at -80C

  • Sigma L4025

  • final concentration 10,000 units/ml in

• Sorb-Trp-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 g/l SC-Trp-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 g/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-Trp-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

15. With cut-tip add 300 ul of spheroplast suspension to sterile 15 ml tubes.

16. With cut-tip add up to 50 ul of ligated YACs or other DNA for each 300 ul of spheroplast suspension

17. Incubate samples for 10 min at room temp. (If DNA is added to cells every 30 sec. then 20 transformations can be done in 10 min.).

18. Add 3 ml of PEG solution and stir carefully resuspend the spheroplasts.

19. Incubate at room temp for 5-10 min.

20. Centrifuge at 22°C and 200-300g for 5 minutes.

21. Remove and discard s/n without loosing the spheroplasts (better with pipette).

22. With cut-tip add 1 ml of SOS and gently resuspend by slow pipetting.

23. Incubate at 30°C without shaking for 30 minutes.

24. Plate spheroplasts as follows:

A) Standard platting: 1. Invert the tube once and add 5 ml (12 ml if 15 cm plates are used) of molten top agar. 2. Close the tube and mix twice by inversion. 3. Pour onto a 37°C pre-warmed sorb-containing plates 4. Tilt the plate to distribute evenly the agar. 5. Incubate upright for at least 15 min. at room temp. 6. Grow at 30°C for 5-6 days (preventing drying after the 2nd day).

B) Platting facilitating “colony breakthrough”: 1. Invert the tube once and add 10.5 ml (with 10 ml plastic pipette) of molten top agar. 2. Close the tube and mix twice by inversion. 3. With the same pipette used in step 1 distribute the spheroplasts-agar onto a big (15 cm) sorb-containing plates pre-warmed to 50°C. 4. Tilt the plate to distribute evenly the agar (this is critical). 5. Incubate upright for at least 15 min. at room temp. 6. Grow at 30°C for 5-6 days (preventing drying after the 2nd day).