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2.8. Protoplast transformation
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A total 1.4 x 106 protoplast cells per sample were resuspended in MMM solution (0.5 M mannitol, 15 mM MgCl2, 0.1% MES). 20 µg of supercoiled plasmid was added to the protoplasts together with PEG solution (0.4 M mannitol, 0.1 M Ca(NO3)2, 40% polyethylene glycol (PEG 4000)) and incubated at room temperature for 1 h. Then, the protoplasts were washed three times with TEX buffer, resuspended in 2 mL TEX after the last centrifugation and incubated in darkness at 26°C for 16 h.
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