Navigation Links
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. In cubation for 10 to 20 min. on ice is possible but not required.
  5. Plate on selective plates (freshly poured top layer).


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Ustilago maydis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/9/2016)... ... 09, 2016 , ... With a presidential election in November and the future ... bring together over 500 top healthcare leaders for a night and day of debates ... by MBA students of the University of Pennsylvania’s Wharton School, will be held February ...
(Date:2/9/2016)... ... 2016 , ... Tunnell Consulting, Inc. announced that Frédéric Lefebvre has joined the ... acquiring new accounts and work closely with existing Tunnell clients throughout Europe to ensure ... European clients more than 15 years of experience in the pharmaceutical industry, a thorough ...
(Date:2/9/2016)... NEW YORK , Feb. 9, 2016 ... market analyzes the current and future prospects of the ... of this report include companies engaged in the manufacture ... comprises an executive summary with a market snapshot providing ... the scope of this report. This section also provides ...
(Date:2/8/2016)... - BIOREM Inc. (TSXV: BRM) ("Biorem" or "the Company") today ... technology companies in the TSX Venture 50 TM . ... the TSX Venture Exchange, in each of five major industry ... & life sciences, diversified industries and technology – based on ... investment, market cap growth, trading volume and analyst coverage. All ...
Breaking Biology Technology:
(Date:2/4/2016)... 4, 2016 The field of Human ... the most popular hubs of the biotechnology industry. ... huge studies of human microbiota, have garnered a ... the microbiome space has literally exploded in terms ... This report focuses on biomedical aspects of research, ...
(Date:2/3/2016)... http://www.researchandmarkets.com/research/d8zjcd/emotion_detection ) ... "Emotion Detection and Recognition Market by Technology ... Tools (Facial Expression, Voice Recognition and Others), ... Global forecast to 2020" report to ... ) has announced the addition of the ...
(Date:2/2/2016)... 2, 2016 Technology Enhancements Accelerate Growth of X-ray ... the digital and computed radiography markets in ... Indonesia (TIM). It provides an ... as well as regional market drivers and restraints. The ... penetration and market attractiveness, both for digital and computed ...
Breaking Biology News(10 mins):