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:5/27/2016)... At present, the Biotech sphere is in a ... volatility is what makes this industry interesting to consider. Here ... (NASDAQ: SNTA ), CTI BioPharma Corp. (NASDAQ: ... and Heat Biologics Inc. (NASDAQ: HTBX ). Sign ... these stocks at: http://www.activewallst.com/register/ ...
(Date:5/26/2016)... Despite the volatility that continues to envelop the ... research on ActiveWallSt.com directs the investor community,s focus on the ... ), Cerus Corp. (NASDAQ: CERS ), Arrowhead Pharmaceuticals ... Inc. (NASDAQ: FPRX ). Register with us today ... On Wednesday, shares in Massachusetts ...
(Date:5/25/2016)... ... May 25, 2016 , ... Lajollacooks4u has become a rising hotspot for specialized ... of its top attractions. Fortune 500 companies, such as Illumina, Hewlett-Packard, Qualcomm and ... intimate team-building experience. , Each event kicks off with an olive oil and salt-tasting ...
(Date:5/25/2016)... ... May 25, 2016 , ... The ... by the Office of the National Coordinator for Health IT (ONC) outlining a ... clinically relevant data were available when and where it was needed. The organization ...
Breaking Biology Technology:
(Date:4/15/2016)... 2016 Research and Markets has ... Market 2016-2020,"  report to their offering.  , ... ,The global gait biometrics market is expected to ... period 2016-2020. Gait analysis generates multiple ... used to compute factors that are not or ...
(Date:3/31/2016)... , March 31, 2016  Genomics firm Nabsys ... founding CEO, Barrett Bready , M.D., who returned ... of the original technical leadership team, including Chief Technology ... of Product Development, Steve Nurnberg and Vice President of ... to the company. Dr. Bready served as ...
(Date:3/22/2016)... PROVO and SANDY, Utah ... Ontario (NSO), which operates the highest sample volume laboratory ... and Tute Genomics and UNIConnect, leaders in clinical sequencing ... announced the launch of a project to establish the ... panel. NSO has been contracted by ...
Breaking Biology News(10 mins):