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Neurospora crassa

Multiporator Transformation Protocol Protocol No. 4308 915.047 11/2001 Organism Neurospora crassa Cell type Filamentous fungus, protoplasts Transformation with Linearized plasmid pKSbar Growth medium Vogels minimal medium, 2% sucrose Washing solution 1 M sorbitol; 1 M sorbitol, 1% PEG 4000 Electroporation buffer 1 M sorbitol, 1% PEG Outgrowth medium Vogels minimal medium, 2% sucrose, 2% sorbose, 1 M sorbitol, 1.2 g/l basta, 2% agar Cuvette Eppendorf, 2 mm gap width, 400 l Temperature 4 C Reference Dr. Ulrich Schulte Institut fr Biochemie Heinrich-Heine-Universitt D-40225 Dsseldorf
Phone +49 211 811 2020 Fax +49 211 811 5310 e-mail ulrich.schulte@uni-duesseldorf.de

Preparation of protoplasts

  1. Grow hyphae 16 h at 28 C in 250 ml medium to a cell density of 25 g/l (wet weight).
  2. Harvest by filtration.
  3. Suspend 3 g hyphae in 40 ml 1 M sorbitol, 20 mg/ml lysing enzymes (Sigma L-1412); mix gently for 2 h at 28 C and remove residual hyphae by filtration.
  4. Centrifuge suspension for 5 min. at 500 x g at 4 C.
  5. Wash twice with 1 M ice-cold sorbitol and one time with 1 M ice-cold sorbitol / 1% PEG.
  6. Resuspend the protoplasts in 0.5 ml ice-cold electroporation buffer (2 x 108 protoplasts/ml), chill on ice for 10 minutes.

Electroporation of protoplasts

  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) 400 V Time constant (T) 500 s No. of pulses (n) 1
  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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