From Lisa Vaillancourt Lab
1. Prepare PEG solution and place on a rocker to mix.
2. Thaw protoplasts on ice.
3. Place 100 mL (10^7 protoplasts) in a pre chilled 50 mL centrifuge tube.
4. Add 3 ug of linearized plasmid pCB1636 and mix gently. DO NOT PIPETTE. Just mix with the tip. (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. Add 1 mL PEG solution dropwise. Mix gently during and after addition.
8. Incubate at room temperature 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). for 200 mL of REG media add 130 uL of Hygromycin.
9. Add 40 mL of regeneration medium equilibrated to 48°C (be careful that the medium is no hotter than 48 °C), replace cap and invert gently 8-10 times to mix.
10. Pour into two Petri plates like 20 mL each. (work fast, media will solidify). Bubbles are OK.
11. Incubate under the lights for about 5-7 days (upside-down)
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 Ingredient final concentration
68.4 g sucrose 1M
3 g bacto agar 1.5%
0.2 g Yeast Extract 0.1%
0.1 g casein hydrolysate (enzymatic) 0.05%
0.1 g casein hydrolysate (acid) 0.05%
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.
130 Î¼L Hygromycin B (383 mg/mL stock solution) 250 Î¼g/mL
From the protocol of Bob Hanau
Prepare a solution of 60% PEG 3350 (w/v, 60gr /100mL) in water and autoclave. Prepare at least 200mL.
Mix 2 parts PEG with 1 part 3X KTC.
Mix well before use. Store at room temperature.
3X KTC solution, 100 mL: (Prepare at least 500 mL)
amount Ingredient Final concentration
13.42 g KCl 1.8 M
15 mL 1M Tris-Cl 150 mM
2.2 g CaCl2x2 H2O 150 mM
Adjust pH to 8.0 and filter sterilize.