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Neurospora crassa

th 1 M ice-cold sorbitol and one time with 1 M ice-cold sorbitol / 1% PEG.
  • Resuspend the protoplasts in 0.5 ml ice-cold electroporation buffer (2 x 108 protoplasts/ml), chill on ice for 10 minutes.
  • Electroporation of cells

    1. Add 4 l plasmid DNA (0.5 mg/ml) to 400 l of protoplasts. Homogenize by gently mixing with pipette several times. Transfer mixture into a pre-chilled cuvette.
    2. Wipe moisture from the cuvette and insert the cuvette into the device.
    3. Electroporation:

      Mode Eukaryotes Voltage (V) 750 V Time constant (T) 250 s No. of pulses (n) 3
    4. Incubation for 10 minutes on ice.
    5. Incubation for 1 h at 28 C in 1 ml outgrowth medium with shaking.
    6. Mix cells with 10 ml Vogels minimal medium, 1 M sorbitol, 2% sorbose, 2% sucrose, 0.7% LMP agarose; plate on selection plates. Incubate 3 days at 28 C.

    Expected result:

    Transformation efficiency up to 100 transformants / g of DNA.


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