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 µl 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 10⁸protoplasts/ml). Keep on ice.

Electroporation of cells:

Add 1-2 µl pNEBUH DNA or 10-20 µl pSMUT DNA (1 µg/µl) to 400 µl of protoplasts. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
Wipe moisture from the cuvette and insert the cuvette into the device.

Electroporation:

Mode	Prokaryotes “O“
Voltage (V)	480 V
Time constant (T)	5 ms

Incubation for 10 to 20 min. on ice is possible but not required.
Plate on selective plates (freshly poured top layer).

Contact Information

In the United States:
Eppendorf North America, Inc.
102 Motor Parkway,
Hauppauge, NY 11788-5178
Tel: 800-645-3050
Fax: 516-334-7506
Web Site: http://www.eppendorfna.com/

Outside the United States:
Eppendorf AG
Barkhausenweg 1
22339 Hamburg
Germany

Customer Service: ++ 49 40 53 801-0

Fax Number: ++ 49 40 53 801-556

Web Site: http://www.eppendorf.com

Ustilago Maydis

Ustilago maydis

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