( ...)Ustilago maydis

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.536 01/2002 Microorganism Ustilago maydis fbd11, protoplasts Cell type Phytopathogenic fungus, yeast-like growing sporidia (diploid) Molecules injected Plasmid DNA (pNEBUH, pSMUT) Growth medium YEPS (1% (w/v) yeast extract, 2% (w/v) bacto-tryptone, 2% (w/v) saccharose) Washing solution SCS (1 M sorbitol, 20 mM sodium citrate (pH 5.8)); 1 M sorbitol Electroporation solution 1 M sorbitol Outgrowth medium 1.5% agar in YEPS with 1 M sorbitol in 94 mm diameter plastic petri dishes
- 10 ml bottom layer containing 300 l hygromycin B/ml agar
- 10 ml top layer of agar (without hygromycin B) poured off 10 to 20 min. prior to use Cuvette 2 mm gap width Reference Robert Fischer Department of Biology Genetics Philipps University of Marburg
Karl-von-Frisch-Str. 1 D-35032 Marburg
Phone +49 6421 282 7080 Fax +49 6421 282 8971 e-mail: rfischer@mailer.uni-marburg.de Making electrocompetent cells:

1. Inoculate 50 ml of YEPS with a primary culture of Ustilago maydis and grow cells overnight at 30 C to a cell density of O.D.₆₀₀ of 0.5 to 0.7. 2. Harvest by centrifugation at 3,000 rpm for 5 min at room temperature. 3. Wash one time with 25 ml SCS. 4. Digest the cell walls by resuspending cells in 2 ml filter sterilized SCS containing 3 mg/ml Novozyme-234, incubate until 20 to 50% of protoplasts have been formed (observe by microscope), then add 25 ml 1 M ice-cold sorbitol. 5. Centrifuge for 7 min at 2,300 rpm at 4 C. 6. Wash three times with 25 ml ice-cold 1 M sorbitol. Centrifuge for 7 min at 2,300 rpm at 4 C. 7. Resuspend in 1 ml ice-cold 1 M sorbitol (density: 10⁷ to 10⁸ protoplasts/ml). Keep on ice.

Electroporation of cells:

1. Add 1-2 l pNEBUH DNA or 10-20 l pSMUT DNA (1 g/l) to 400 l of protoplasts. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
2. Wipe moisture from the cuvette and insert the cuvette into the device.
3. Electroporation:
   
   Mode Prokaryotes O Voltage (V) 480 V Time constant (T) 5 ms
   
4. Incubation for 10 to 20 min. on ice is possible but not required.
5. Plate on selective plates (freshly poured top layer).
   
   

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1. Ustilago maydis