Navigation Links
721.82

Multiporator Transfection Protocol Protocol No. 4308 915.045 11/2001 Cell line 721.82, EBV transfected B cell line, MHC class I positive,
MHC class II DR, DQ negative Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Kirsten Falk and Dr. Olaf Rtzschke Cellular Immunology
Max Delbrck Center for Molecular Medicine Robert-Rssle-Strasse 10 D-13125 Berlin-Buch
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells and centrifuge them (for 5 minutes, 200 x g, at room temperature). Remove supernatant.
  3. Resuspend the cells in medium, set the cell number to 1 x 106 cells/ml and centrifuge them (for 5 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!

  4. Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and mix plasmid DNA (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) 200 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 15 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 3 ml RPMI /10% FCS, and cultivate them in a 55 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pEGFP-N1 by viable 721.82 cells can be detected after 24 hours with a flow cytometer by costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT. Result: Survival rate: 67.6% Transfection rate: 12.9% based on the number of surviving cells. Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. 721.221
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/9/2017)... ... October 09, 2017 , ... At its national board meeting in North ... in Harvard University’s Departments of Physics and Astronomy, has been selected for membership in ... winning team for the 2015 Breakthrough Prize in Fundamental physics for the discovery of ...
(Date:10/7/2017)... ... October 06, 2017 , ... ... consulting for microscopy and surface analysis, Nanoscience Instruments is now expanding into ... a broad range of contract analysis services for advanced applications. Services will ...
(Date:10/6/2017)... ... October 06, 2017 , ... The HealthTech Venture Network ... at their fourth annual Conference where founders, investors, innovative practitioners and collaborators are ... pitch competition showcasing early stage digital health and med tech companies. , This ...
(Date:10/5/2017)... ... 2017 , ... Understanding the microbiome, the millions of bacteria that live in ... Are My Future, the newest exhibit on display at the University City Science Center’s ... through the lens of the gut microbiome. , Gut Love opens October 12, ...
Breaking Biology Technology:
(Date:8/15/2017)...   ivWatch LLC , a medical device company focused on ... receipt of its ISO 13485 Certification, the global standard for medical ... Standardization (ISO®). ... Continuous Monitoring device for the early detection of IV infiltrations. ... "This is an important milestone for ivWatch, as ...
(Date:6/30/2017)... ARLINGTON, Va. , June 30, 2017 /PRNewswire-USNewswire/ ... a leading developer and supplier of face and ... the ATA Featured Product provider program. ... created an innovative way to monitor a driver,s ... benefit greatly from being able to detect fatigue ...
(Date:6/14/2017)... 15, 2017  IBM (NYSE: IBM ) is introducing several ... dedicated to developing collaboration between startups and global businesses, taking ... During the event, nine startups will showcase the solutions they ... industries. France is ... with a 30 percent increase in the number of startups ...
Breaking Biology News(10 mins):