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:3/22/2017)... , March 22, 2017 Good Start ... it has eclipsed the 130 million covered lives mark ... Shield of Texas . With newly ... Company continues to enjoy strong payor acceptance based on ... clinical programs and genetic counseling, its industry-leading customer care ...
(Date:3/22/2017)... New York , March 22, 2017 ... is largely fragmented, states a research report by Transparency ... S.A., Pfizer Inc., Amgen Inc., and AbbVie Inc., accounted ... in 2015. The prominent players in this market are ... expand their product portfolio, which is likely to lead ...
(Date:3/22/2017)... , March 22, 2017   Boston Biomedical , ... therapeutics designed to target cancer stemness pathways, today announced ... Andrews as Chief Executive Officer, effective April 24, ... Chiang J. Li , M.D., FACP, who has led ... ago. Under his leadership, Boston Biomedical has grown from ...
(Date:3/22/2017)... ... March 21, 2017 , ... Okyanos Cell Therapy has announced Tallahassee, FL ... live events series, “Stem Cell Therapy: The Next Phase in the Evolution of Medicine.” ... Stem Cell Research and Therapy Act, Okyanos maintains a mission to help “no-option” ...
Breaking Biology Technology:
(Date:3/2/2017)... LONDON , March 2, 2017 Summary ... require to better understand Merck KGaA and its partnering ... report: https://www.reportbuyer.com/product/3605601/ Description The Partnering Deals ... into the partnering activity of one of the world,s ... reports are prepared upon purchase to ensure inclusion of ...
(Date:2/28/2017)... LOS ANGELES , Feb. 28, 2017   ... identity verification software globally, announces significant enhancements to new ... in May 2016. New products include mobile and desktop ... and DocX TM - a real time manual ... Acuant,s core idScan® technology provides the fastest and most ...
(Date:2/26/2017)... , Feb. 25, 2017  Securus Technologies, ... technology solutions for public safety, investigation, corrections and ... Recidivism and Reentry. "Too often, too ... prisons and county jails are trying to tackle ... inmates and friends and family members. While significant steps ...
Breaking Biology News(10 mins):