Navigation Links
Jurkat

Multiporator Transfection Protocol Protocol No. 4308 915.020 11/1999 Cell line Jurkat, T-lymphocyte, human leukemia (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Prof. Ulrich Zimmermann Lehrstuhl fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508 Fax +49 931 888 4509
e-mail: zimmerma@biozentrum.uni-wuerzburg.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in RPMI 1640 / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 minutes, 200 x g, at room temperature). Remove supernatant.

    Note: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to guarantee a successful electroporation!

  3. Resuspend the cells in Hypoosmolar Electroporation Bu ffer. When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid DNA (5-20 g/ml final concentration, in bidistilled H2O).
  5. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 240 V Time constant (T) 40 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 5 ml RPMI 1640 / 10% FCS, and cultivate it in a 60 mm culture dish.
    Note: After pulsing, the cells should be incubated for 2-3 h at 37 C before any centrifugation is performed, to ensure resealing of the membrane.
Detection methods for transfection:
The expression of the plasmid pEGFP-N1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 85% Transfection rate: 67% based on the number of surviving cells
57% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Kluyveromyces lactis
2. Lactococcus lactis
3. Lactococcus lactis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/4/2016)... LEXINGTON, Massachusetts , February 4, 2016 - New ... --> - New FDA action date of July ... date of July 22, 2016   - ... the U.S. in the past decade indicated for the treatment of signs and ... Lifitegrast has the potential to be the only product approved in the ...
(Date:2/4/2016)... , Feb. 4, 2016 Beike Biotechnology, ... various medical institutions attended a ceremony in late 2015 ... personalized cell therapy in 2016. --> ... Clinical Translation Platform for Personalized Cell Therapy" was hosted ... Cell Production Center, both subsidiaries of Beike Biotechnology Co., ...
(Date:2/4/2016)... N.J. , Feb. 4, 2016 ContraVir ... focused on the development and commercialization of targeted antiviral ... CEO & Investor Conference 2016, to be held February ... Source Capital Group,s 2016 Disruptive Growth & Healthcare Conference, ... on February 10-11, 2016. James Sapirstein , ...
(Date:2/4/2016)... England , February 4, 2016 ... Bioscience Laboratories (ABL), Inc. --> Strasbourg, France ... Inc. --> PharmaVentures is pleased to announce that ... of its biopharmaceutical manufacturing unit in Strasbourg, France ... Inc. --> --> Transgene ...
Breaking Biology Technology:
(Date:2/3/2016)... -- Vigilant Solutions announces today that the ... solved two recent hit-and-run cases with the ... Solutions. Brian Wenberg explains, "I was ... out of a convenience store and witnessed an elderly male back out ... his vehicle and leaving the scene.  In his statement ...
(Date:2/2/2016)... NEW YORK , Feb. 2, 2016 Technology ... service presents an analysis of the digital and computed ... Malaysia , and Indonesia ... current trends and market size, as well as regional ... by country and discusses market penetration and market attractiveness, ...
(Date:2/1/2016)... -- Wocket® smart wallet ( www.wocketwallet.com ) announces the launch of a new ... Las Vegas , where Joey appeared at the ... Las Vegas , where Joey appeared at the Wocket booth to ... was filmed at the Consumer Electronics Show (CES2016) in Las ... and greet fans. --> --> ...
Breaking Biology News(10 mins):