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:6/24/2016)... 2016 Epic Sciences unveiled a liquid ... to PARP inhibitors by targeting homologous recombination deficiency ... new test has already been incorporated into numerous ... types. Over 230 clinical trials are ... including PARP, ATM, ATR, DNA-PK and WEE-1. Drugs ...
(Date:6/24/2016)... ... ... Researchers at the Universita Politecnica delle Marche in Ancona combed medical journal articles ... findings are the subject of a new article on the Surviving Mesothelioma website. ... blood, lung fluid or tissue of mesothelioma patients that can help point doctors to ...
(Date:6/23/2016)... WI (PRWEB) , ... June 23, 2016 , ... ... supplements, is pleased to announce the launch of their brand, UP4™ Probiotics, into ... for over 35 years, is proud to add Target to its list of ...
(Date:6/23/2016)... 23, 2016 /PRNewswire/ - FACIT has announced the ... biotechnology company, Propellon Therapeutics Inc. ("Propellon" or ... of a portfolio of first-in-class WDR5 inhibitors for ... as WDR5 represent an exciting class of therapies, ... medicine for cancer patients. Substantial advances have been ...
Breaking Biology Technology:
(Date:4/15/2016)... , April 15, 2016 ... the,  "Global Gait Biometrics Market 2016-2020,"  report to ... http://photos.prnewswire.com/prnh/20160330/349511LOGO ) , ,The global gait biometrics ... of 13.98% during the period 2016-2020. ... angles, which can be used to compute factors ...
(Date:3/31/2016)...  Genomics firm Nabsys has completed a financial  restructuring ... , M.D., who returned to the company in October ... team, including Chief Technology Officer, John Oliver , ... and Vice President of Software and Informatics, Michael ... Dr. Bready served as CEO of Nabsys from 2005-2014 ...
(Date:3/22/2016)... , PROVO and ... Newborn Screening Ontario (NSO), which operates the highest sample ... molecular testing, and Tute Genomics and UNIConnect, leaders in ... respectively, today announced the launch of a project to ... (NGS) testing panel. NSO has been ...
Breaking Biology News(10 mins):