Plasmid delivery using electroporation

Adapted from The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous URA3 as a Reporter Gene in Ganoderma lucidum, Efficient Transformation of the Medicinal Mushroom Ganoderma lucidum, Smolke Lab Yeast Transformation, Yeast Electroporation

1. Inoculate a starter plate made from either PDYA or MPDYA with a single 5mm plug of tissue from a mother plate. Culture at 30C for 3-4 days.
2. Inoculate 50 mL of PDY or MPDY in a 125 mL flask with 5mm plugs (9x) from leading edge tissue from the starter plate culture. Culture at 30C, 70 rpm for 5-7 days.
3. Collect mycelia in cell strainer, discarding left over pieces of agar.
4. Wash mycelia in sorbitol, spin down, discard supernatant
5. Collect mycelia and wash in KCl/NaP, spin down or collect in cell strainer
6. Incubate 15mL enzyme solution (.2 g enzyme in 5 mL KCl/NaP) at 37C for 15 min
7. Add 15mL KCl/NaP to mycelia in 250 mL flask
8. Add activated and filtered enzyme solution (15mL) to mycelia mix in flask. incubate mix for 1.5 hours
9. Spin down digested mycelia and discard supernatant
10. Resuspend mycelia in 20 mL ice cold sterile water. spin down, discard supernatant
11. Treat cells with 20mL LiAc solution at 30C for 30 min
12. Add 0.2mL DTT solution to mix and incubate at 30C for 15 min
13. Use T. Chalmers and Gothenburg protocol from here on out.
14. Re-suspend cells with 1mL of sterilized ice-cold 1 M sorbitol.
15. Take 200 μl of suspended cells into a new 1.5 ml tube on ice.
16. Add 15 μl fragment/plasmid DNA (> 500 ng/μl). Mix with pipetman and keep on ice for 15 min. Transfer all to a sterilized cuvette (green cap). Add 1 ml of cold 1 M sorbitol to new labeled tubes (used later).
17. Set the cuvette in the holder of Micro Pulser Electroporator. Chose “Manual”, and set voltage at 1.5 kV. Push the pulse button. Read “Time / ms”, if it is between 4.0-6.0, this process is successful.
18. Add 1 ml of cold 1 M sorbitol, immediately after the pulse. Mix well by pipetting up and down. (After this, it is OK to be at RT.) Transfer all to the sorbitol tube ASAP.
19. Incubate at 30°C for 1-3 h. Centrifuge (3000 g, 1 min) to ~150 μl and then spread cells on selection plates. Make a negative control plate. Place the plates at 30°C air incubator.