7. MgCl 2 .
8. Ca(NO 3 ) 2 .
9. β-mercaptoethanol.
10. PEG6000.
11. Nylon filter mesh, 60 micron (Fisher Scientific).
12. 5 mL pipettes.
13. Round bottom polystyrene tubes, 14 mL.
14. 500-mL sterile side-arm flask.
15. Double-sided thin razor blades.
16. 24-well flat bottomed microplate for fluorescent microscopy.
17. 500 μL tips with a cut end to widen the opening (cut with a
razor blade or scissors and autoclave).
18. Vermiculite.
19. Perlite.
20. Fluorescent microscope (e.g., Leica Microsystems) equipped
with lasers or lamp filters for detection of the fluorescent signal
of choice.
21. Buffer A. Basic protoplast buffer: 10 mM CaCl 2 , 0.6 M mannitol, 20 mM MES-KOH (pH 5.7), autoclaved and stored at
room temperature.
22. Buffer B. Isolation buffer: to 50 mL of Buffer A, add 1%
cellulase (500 mg), 0.3% pectinase (160 mg), 5 mM (18 μL)
β-mercaptoethanol, and 0.1% BSA (50 mg). Alternatively, use
300–500 mg of Onozuka R10 mix, containing cellulase and
pectinase). This buffer must be made immediately before use.
Keep at room temperature. Do not store.
23. Buffer C. PEG solution: in a 50 mL plastic tube mix 6 mL of a
0.1 M Ca(NO 3 ) 2 solution with 0.91 g mannitol (0.5 M final
concentration). Dissolve the mannitol by swirling, then add 4 g
of PEG6000 (40% final concentration). Tightly close the tube
and microwave on high (~800 watts) for exactly 6 s so that the
solution does not boil and change concentration. Gently mix,
microwave again for 6 s, and gently mix again. Let the solution
cool down. The solution must appear clear with no visible
particles. This solution must be prepared fresh before use and
cooled down to room temperature. Do not store.
24. Buffer D. Incubation buffer: 15 mM MgCl 2 , 0.5 M mannitol,
5 mM MES-KOH (pH 5.5), autoclaved and stored at room
temperature.
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Maria Shumskaya et al.
8. Ca(NO 3 ) 2 .
9. β-mercaptoethanol.
10. PEG6000.
11. Nylon filter mesh, 60 micron (Fisher Scientific).
12. 5 mL pipettes.
13. Round bottom polystyrene tubes, 14 mL.
14. 500-mL sterile side-arm flask.
15. Double-sided thin razor blades.
16. 24-well flat bottomed microplate for fluorescent microscopy.
17. 500 μL tips with a cut end to widen the opening (cut with a
razor blade or scissors and autoclave).
18. Vermiculite.
19. Perlite.
20. Fluorescent microscope (e.g., Leica Microsystems) equipped
with lasers or lamp filters for detection of the fluorescent signal
of choice.
21. Buffer A. Basic protoplast buffer: 10 mM CaCl 2 , 0.6 M mannitol, 20 mM MES-KOH (pH 5.7), autoclaved and stored at
room temperature.
22. Buffer B. Isolation buffer: to 50 mL of Buffer A, add 1%
cellulase (500 mg), 0.3% pectinase (160 mg), 5 mM (18 μL)
β-mercaptoethanol, and 0.1% BSA (50 mg). Alternatively, use
300–500 mg of Onozuka R10 mix, containing cellulase and
pectinase). This buffer must be made immediately before use.
Keep at room temperature. Do not store.
23. Buffer C. PEG solution: in a 50 mL plastic tube mix 6 mL of a
0.1 M Ca(NO 3 ) 2 solution with 0.91 g mannitol (0.5 M final
concentration). Dissolve the mannitol by swirling, then add 4 g
of PEG6000 (40% final concentration). Tightly close the tube
and microwave on high (~800 watts) for exactly 6 s so that the
solution does not boil and change concentration. Gently mix,
microwave again for 6 s, and gently mix again. Let the solution
cool down. The solution must appear clear with no visible
particles. This solution must be prepared fresh before use and
cooled down to room temperature. Do not store.
24. Buffer D. Incubation buffer: 15 mM MgCl 2 , 0.5 M mannitol,
5 mM MES-KOH (pH 5.5), autoclaved and stored at room
temperature.
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Maria Shumskaya et al.