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:5/25/2016)... ... May 25, 2016 , ... The Ankle Plating System ... designed to address fractures of the distal tibia and fibula. This system marks ... Plating System 3 is composed of seven plate families that span the lateral, ...
(Date:5/25/2016)... ... 2016 , ... The American Medical Informatics Association (AMIA) responded ... Coordinator for Health IT (ONC) outlining a measurement approach to interoperability that focuses ... and where it was needed. The organization of health informatics professionals said a ...
(Date:5/25/2016)... ... 25, 2016 , ... WEDI, the nation’s leading authority on the use of ... Stellar has been named by the WEDI Board of Directors as WEDI’s president and ... leader with more than 35 years of experience in healthcare, association management and organizational ...
(Date:5/25/2016)... ... 25, 2016 , ... Biohaven Pharmaceutical Holding Company Ltd. (Biohaven) ... company’s orphan drug designation request covering BHV-4157 for the treatment of Spinocerebellar Ataxia ... , Spinocerebellar ataxia is a rare, debilitating neurodegenerative disorder that is estimated ...
Breaking Biology Technology:
(Date:3/21/2016)... , March 22, 2016 ... with passcodes for superior security   ... leading provider of secure digital communications services, today announced ... technology and offer enterprise customers, particularly those in the ... recognition and voice authentication within a mobile app, alongside, ...
(Date:3/15/2016)... JERUSALEM , March 15, 2016 ... Jerusalem , the technology-transfer company of the Hebrew University, ... developer of remote sensing technology of various human biological ... funding, raising $2.0 million from private investors. ... technology, based on the detection of electromagnetic emissions from ...
(Date:3/11/2016)... March 11, 2016 --> ... research report "Image Recognition Market by Technology (Pattern Recognition), ... Advertising), by Deployment Type (On-Premises and Cloud), by Industry ... published by MarketsandMarkets, the global market is expected to ... 29.98 Billion by 2020, at a CAGR of 19.1%. ...
Breaking Biology News(10 mins):