1. Prepare PEG solution and place on a rocker to mix.
2. Thaw protoplasts on ice.
3. Place 100 ml (107 protoplasts) in a pre chilled 50 ml centrifuge tube.
4. Add 3 mg of linearized plasmid pCB1636 and mix gently. (Note: you can also use circular plasmid, but the transformation efficiency will be quite a lot lower).
5. Incubate on ice 20 minutes.
6. Place tube in a rack at room temperature. (Note: for REMI add 20-40 units of the appropriate restriction enyme and gently mix.)
7. Drop-wise, add 1 ml PEG solution. Mix gently during and after addition.
8. Incubate at room temp for 20 minutes. Meanwhile, Add hygromycin to the regeneration medium. (Note: to transform to phleomycin resistance, do not add hygromycin, instead add 100 ml of phleomycin to each 40 ml tube of regeneration medium, and mix gently).
9. Add 40 ml of regeneration medium equilibrated to 48C (be careful that the medium is no hotter than 48!), replace cap and invert gently 8-10 times to mix.
10. Pour into two Petri plates.
11. Incubate under the lights for about 5-7 days.
12. Transfer transformants to PDA plates + 50 mg/ml Hygromycin B and incubate under lights for about 1.5-2 weeks. I usually use 24 well culture dishes with 2 ml medium per well. Transformation efficiency has been around 10-100 transformants per mg DNA.
Regeneration medium 200 mL (From the protocol of Bob Hanau)
amount final conc. ingredient
68.4 g 1M sucrose
3 g 1.5% agar
0.2 g 0.1% Yeast Extract
0.1 g 0.05% casein hydrolysate (enzymatic)
0.1 g 0.05% casein hydrolysate (acid)
160 mL H2O
To avoid boilovers in the autoclave, prepare in a 1 L Erlenmyer flask and completely dissolve the sucrose before autoclaving. Add Hygromycin just before using the medium
128 mL 250 mg/mL Hygromycin B (390mg/mL stock solution)
PEG solution (From the protocol of Bob Hanau)
Prepare a solution of 60% PEG 3350 (w/v) in water and autoclave.
Mix 2 parts PEG with 1 part 3X KTC. Mix well before use.
3X KTC solution, 100 ml:
amount final conc. ingredient
13.42 g 1.8 M KCl
15 ml 150 mM 1M Tris-Cl
2.2 g 150 mM CaCl2€2 H2O
Adjust pH to 8.0 and filter sterilize.
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