Linear fragment delivery using PEG-mediated transformation

I intended this protocol to be used for transient expression experiments using linear amplicons of expression cassettes.

1. Inoculate 50 mL PDY or MPDY in a 125 mL flask with 9 plugs of leading edge tissue. Culture at 30C, 70 rpm for 5 days.
2. Prepare 20ml of lysing enzymes (0.04g/ml of Lysing Enzyme from Trichoderma harzanium in 0.6 M KCl, 0.1 M phosphate buffer pH 5.8) and incubate 15min at 37C, 200 rpm (use a 50 ml tube)
3. Collect 5 day old mycelium from the flask using a 40um filter and wash it using 10ml of 0.6 M KCl, 0.1 M phosphate buffer pH 5.8 (use a 50 ml tube and 40 um filter).
   1. A second wash can sometimes improve yield with older cultures: resuspend the mycelia in 10ml of 0.6 M KCl, 0.1 M phosphate buffer pH 5.8 in a 50 mL conical tube, vortex, and then centrifuge at 6k rpm for 5 minutes. Discard the supernatant.
4. Transfer the mycelium into a single 125 ml flask,
5. Filter lysing enzymes (0.2um syringe filter) and add the enzyme to the flask with mycelia. Incubate for 3-4h, 25C, 70 rpm.
6. Collect 10 µL and check for protoplast formation.
7. Filter the protoplasts through sterile cheese cloth stuffed into a 60 mL syringe that is directed into a 40um cell filter over a 50 mL conical tube.
   1. If needed filter through a 5 um MILLEX syringe filter into a clean conical tube.
8. Collect the protoplasts by centrifugation, 3500 rpm, 4C
9. Discard the supernatant and wash the protoplasts using 10ml of TMS
10. Collect the protoplasts by centrifugation, 3500 rpm, 4C
11. Discard the supernatant and resuspend the protoplast into 300ul of TMSC
12. Count the protoplasts
    1. Using microscope at 40x and Neubauer improved 0.1mm 0.0025 mm^2
    2. Image all four large corner squares each composed of 16 small squares.
    3. Using Fiji count protoplasts in all 64 squares.
    4. http://insilico.ehu.eus/counting_chamber/neubauer_improved.php
    5. https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx
13. Adjust the protoplast to 2.10^7 protoplasts / 100ul
14. Prepare the DNA solution :
    1. 2ug of PCR product into 30ul of H2O + 30 ul of TE CaCl2 2x (optional: add 5µL of single stranded Salmon Sperm DNA as carrier DNA)
15. Add the 60ul DNA solution into the 100ul of protoplasts and incubate on ice for 20 minutes.
16. Dilute 1.2g of PEG 6000 into 800ul MS (per sample), microwave approximately 1min, until mix becomes clear and contains no precipitates (do not overheat, mix will turn cloudy), then cool down at RT
17. Add the 800ul PEG into the 160ul protoplast + DNA
18. Mix gently and incubate 15min at RT
19. Add 1ml of TMSC into the PEG-protoplast solution
20. Centrifuge 5min at 5000rpm (minispin)
21. Discard the supernatant and resuspend the pellet into 400ul of TMSC
22. Add 100 µL of PDY or MPDY to the 400 ul of protoplast solution and incubate on the barrel rotator in the 30C incubator for at least four hours.
    1. Incubation times and conditions can be adjusted depending on application, e.g. overnight at room temperature.
23. Spin down the sample at 5k rpm for 5 minutes.
24. Resuspend the sample in 100 µL of TMSC or sorbitol.
25. Pipette 10 µL onto a glass slide and cover with a cover slip.
26. Image using an appropriate fluorescent microscope, plate-reader, or proceed with appropriate assay.

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  Representative Brightfield micrograph image of transformed protoplasts after 4 hours of incubation, 20x magnification.

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  Representative FITC micrograph image of transformed protoplasts after 4 hours of incubation, 20x magnification.