Navigation Links
Neurospora crassa

Multiporator Transformation Protocol Protocol No. 4308 915.047 11/2001 Organism Neurospora crassa Cell type Filamentous fungus, protoplasts Transformation with Linearized plasmid pKSbar Growth medium Vogels minimal medium, 2% sucrose 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

Preparation of protoplasts

  1. Grow hyphae 16 h at 28 C in 250 ml medium to a cell density of 25 g/l (wet weight).
  2. Harvest by filtration.
  3. Suspend 3 g hyphae in 40 ml 1 M sorbitol, 20 mg/ml lysing enzymes (Sigma L-1412); mix gently for 2 h at 28 C and remove residual hyphae by filtration.
  4. Centrifuge suspension for 5 min. at 500 x g at 4 C.
  5. Wash twice with 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 protoplasts

  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) 400 V Time constant (T) 500 s No. of pulses (n) 1
  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:2/10/2016)...  IsoRay, Inc. (NYSE MKT: ISR), a medical technology ... applications for the treatment of prostate, brain, lung, head ... results for the second quarter and six months of ... --> --> Revenue was ... which ended December 31, 2015, a 12% increase compared ...
(Date:2/10/2016)... ... February 10, 2016 , ... Benchmark Research, a ... promotion of two long-standing principal investigators (PI) to the roles of Chief Medical ... Development. , Dr. Laurence Chu, a Benchmark Research PI in the Austin office, ...
(Date:2/10/2016)... Charlotte, NC (PRWEB) , ... February 10, 2016 ... ... highly anticipated expansion to their comprehensive training and support program, Sonalinkā„¢ remote monitoring. ... Sonablate® HIFU procedures performed on Friday, February 5th, connecting Dr. Samuel Peretsman to ...
(Date:2/10/2016)... ... 10, 2016 , ... Global Stem Cells Group, ... Global Stem Cells Network (GSCN) and its affiliate Global Medical Training Network ... adipose and bone marrow therapies. , Through the new collaboration, Global Stem ...
Breaking Biology Technology:
(Date:2/2/2016)... Feb. 2, 2016 Checkpoint Inhibitors for ... Market Are you interested in the future ... for checkpoint inhibitors. Visiongain,s report gives those predictions ... and national level. Avoid falling behind in ... opportunities and revenues those emerging cancer therapies can ...
(Date:2/1/2016)... -- Rising sales of consumer electronics coupled ... gesture control market size through ... electronics coupled with new technological advancements to drive global ... through 2020   --> Rising ... to drive global touchfree intuitive gesture control market ...
(Date:1/27/2016)... Ohio , Jan. 27, 2016  Rite Track, ... based in West Chester, Ohio ... award winning service staff, based in Austin, ... capacity and ability to provide modifications, installations and technical ... Dovalina , CEO of PLUS, commented, "PLUS has provided ...
Breaking Biology News(10 mins):