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:2/24/2017)... ... February 24, 2017 , ... FireflySci, Inc is an explosive small business that ... the goal of bringing their powerful cuvette and spectrophotometer calibration to the ... FireflySci is going on as they add yet another mark on the global map. ...
(Date:2/23/2017)... SAN RAFAEL, Calif., Feb. 23, 2017 ... of U.S. dollars, except per share data, unaudited)Three Months ... ChangeTotal BioMarin Revenue $ ...     22832%$ 1,117$   89026%Aldurazyme Net Product Revenue ... 906538%34823946%Naglazyme Net Product Revenue  ...
(Date:2/23/2017)... -- Seattle,s upscale Capitol Hill neighborhood, with its swanky shops, parks and ... lice treatment salon to set up shop. But there,s ... French bistro on E Madison Ave, and CEO Maria ... lice clinic, we pride ourselves on being a destination for ... the stigma associated with lice. Everyone can get lice – ...
(Date:2/23/2017)... ... ... Today, researchers can fast-track sample collection and analysis for ... or SNPs of interest) using one, easy-to-collect saliva sample. With the addition of ... and other relevant biomarkers can be extensively studied through a non-invasive sample type. ...
Breaking Biology Technology:
(Date:2/21/2017)... ARMONK, N.Y. and PORTLAND, Ore. ... IBM ) and the Avamere Family of Companies (Avamere ... Care) today announced a six-month research study that will ... caregivers improve eldercare at senior living and health centers. ... facilities, Avamere hopes to gain insights into physical and ...
(Date:2/14/2017)... WINSTON-SALEM, N.C. , Feb. 14, 2017  Wake ... FRY-shlog), M.D., as its new chief executive officer (CEO). ... succeeds CEO John D. McConnell , M.D., who ... new position at the Medical Center, after leading it ... oversee the full scope of Wake Forest Baptist,s academic ...
(Date:2/10/2017)... , Feb 10, 2017 ... new report "Personalized Medicine - Scientific and Commercial Aspects" ... ... personalized medicine. Diagnosis is integrated with therapy for selection of ... on early detection and prevention of disease in modern medicine. ...
Breaking Biology News(10 mins):