Navigation Links
Neurospora crassa

Multiporator Transformation Protocol Protocol No. 4308 915.048 11/2001 Organism Neurospora crassa Cell type Filamentous fungus, conidia Transformation with Linearized plasmid pKSbar Growth medium Vogels minimal medium, 2% sucrose, 2% agar Washing solution 1 M sorbitol; 1 M sorbitol, 1% PEG 4000 Electroporation buffer 1 M sorbitol, 1% PEG Outgrowth medium Vogels minimal medium, 2% sucrose, 2% sorbose, 1 M sorbitol, 1.2 g/l basta, 2% agar Cuvette Eppendorf, 2 mm gap width, 400 l Temperature 4 C Reference Dr. Ulrich Schulte Institut fr Biochemie Heinrich-Heine-Universitt D-40225 Dsseldorf
Phone +49 211 811 2020 Fax +49 211 811 5310 e-mail ulrich.schulte@uni-duesseldorf.de

Treatment of cells

  1. Grow cells 3-4 days at 28 C on agar-medium (100 ml).
  2. Harvest by suspension in 1 M sorbitol and filtration.
  3. Suspend 8 x 108 conidia/ml in 1 M sorbitol, 20 mg/ml lysing enzymes (Sigma L-1412); mix gently for 2 h at 28 C.
  4. Centrifuge suspension for 5 min. at 500 x g at 4 C.
  5. Wash twice wi th 1 M ice-cold sorbitol and one time with 1 M ice-cold sorbitol / 1% PEG.
  6. Resuspend the protoplasts in 0.5 ml ice-cold electroporation buffer (2 x 108 protoplasts/ml), chill on ice for 10 minutes.

Electroporation of cells

  1. Add 4 l plasmid DNA (0.5 mg/ml) to 400 l of protoplasts. Homogenize by gently mixing with pipette several times. Transfer mixture into a pre-chilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Eukaryotes Voltage (V) 750 V Time constant (T) 250 s No. of pulses (n) 3
  4. Incubation for 10 minutes on ice.
  5. Incubation for 1 h at 28 C in 1 ml outgrowth medium with shaking.
  6. Mix cells with 10 ml Vogels minimal medium, 1 M sorbitol, 2% sorbose, 2% sucrose, 0.7% LMP agarose; plate on selection plates. Incubate 3 days at 28 C.

Expected result:

Transformation efficiency up to 100 transformants / g of DNA.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Efficient Transfection of Neurospora Crassa
2. Efficient Transfection of Neurospora Crassa
3. Neurospora crassa
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... San Diego, CA (PRWEB) , ... June 27, 2016 , ... ... mClinical solutions for clinical trials, announced today the Clinical Reach Virtual Patient ... and their care circle with the physician and clinical trial team. , Using the ...
(Date:6/27/2016)... , June 27, 2016   Ginkgo Bioworks ... to industrial engineering, was today awarded as one ... selection of the world,s most innovative companies. Ginkgo ... scale for the real world in the nutrition, ... engineers work directly with customers including Fortune 500 ...
(Date:6/24/2016)... ... June 24, 2016 , ... Researchers at the Universita Politecnica delle Marche in ... peritoneal or pleural mesothelioma. Their findings are the subject of a new article on ... biomarkers are signposts in the blood, lung fluid or tissue of mesothelioma patients that ...
(Date:6/23/2016)... , June 23, 2016 /PRNewswire/ - FACIT has ... Ontario biotechnology company, Propellon Therapeutics Inc. ... and commercialization of a portfolio of first-in-class WDR5 ... targets such as WDR5 represent an exciting class ... in precision medicine for cancer patients. Substantial advances ...
Breaking Biology Technology:
(Date:6/2/2016)... , June 2, 2016   The Weather Company ... announcing Watson Ads, an industry-first capability in which consumers will ... being able to ask questions via voice or text and ... Marketers have long sought an ... consumer, that can be personal, relevant and valuable; and can ...
(Date:6/1/2016)... NEW YORK , June 1, 2016 ... Biometric Technology in Election Administration and Criminal Identification to ... According to a recently released TechSci Research report, " ... Sector, By Region, Competition Forecast and Opportunities, 2011 - ... $ 24.8 billion by 2021, on account of growing ...
(Date:5/20/2016)... , May 20, 2016  VoiceIt is excited ... with VoicePass. By working together, VoiceIt ...  Because VoiceIt and VoicePass take slightly different approaches ... increases both security and usability. ... about this new partnership. "This marketing ...
Breaking Biology News(10 mins):