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:3/24/2017)... 2017   Sienna Biopharmaceuticals, Inc. , a privately ... that Richard Peterson will join the company ... Peterson, who brings more than two decades of global ... is retiring at the end of April but will ... joins Sienna from Novan, Inc., where he served as ...
(Date:3/23/2017)... , March 23, 2017  Northwest Biotherapeutics ... DCVax® personalized immune therapies for solid tumor cancers, ... $7.5 million financing it announced last Friday, March ... to several institutional investors securities totaling 28,843,692 shares, ... share, and 10,000,000 shares of Class C Warrants ...
(Date:3/23/2017)... 2017 Kineta, Inc., a biotechnology company ... in immuno-oncology, today announced the discovery and characterization ... that activate interferon response factor 3 (IRF3) via ... tumor regression in a murine colon carcinoma mouse ... complete tumor regression to initial drug treatment were ...
(Date:3/23/2017)... ... March 23, 2017 , ... ... announced the hire of Dr. Sigmund “Sig” Floyd as Vice President ? Global ... and joint development activities. , “Dr. Floyd’s career has spanned 30 years in ...
Breaking Biology Technology:
(Date:2/28/2017)... BARCELONA , Spanien, 27. Februar 2017 /PRNewswire/ ... durch Iris-Scan, wird seine erstklassige biometrische Lösung ... Snapdragon™ 835 mit X16 LTE auf dem ... 2. März) am Qualcomm-Stand in Halle 3, ... 835-Prozessor beinhaltet die Sicherheitsplattform Qualcomm Haven™ – ...
(Date:2/24/2017)... -- EyeLock LLC, a leader of iris-based identity authentication ... solution on the latest Qualcomm® Snapdragon™ 835 mobile ... Congress 2017 (February 27 – March 2, ... Stand 3E10. The Snapdragon 835 ... combination of hardware, software and biometrics technologies ...
(Date:2/21/2017)... February 21, 2017 Der weltweite ... wachsen. Nach einem Gespräch mit mehr als 50 Vertretern aus ... zu überwinden gilt, um diese Prognose zu realisieren. ... ... Mobilisierung der finanziellen Mittel für die Biobank, die Implementierung ...
Breaking Biology News(10 mins):