Navigation Links
721.174

Multiporator Transfection Protocol Protocol No. 4308 915.043 11/2001 Cell line 721.174, EBV transfected B cell line, MHC class I positive, MHC class II 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. Add and mix plasmid DNA (12.5 g/ml final concentration, in bidistilled H2O).
  6. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  7. Electroporation:

    Mode Eukaryotes Voltage (V) 200 V Time constant (T) 40 s No. of pulses (n) 1
  8. After the pulse, allow the cell suspension to stand in the cuvette for 15 minutes at room temperature.
  9. 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 721.174 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: 72.54% Transfection rate: 20.77% based on the number of surviving cells. Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Ustilago maydis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... , June 23, 2016  Blueprint Bio, a company ... to the medical community, has closed its Series A ... Nunez . "We have received a commitment ... capital we need to meet our current goals," stated ... us the runway to complete validation on the current ...
(Date:6/23/2016)... , June 23, 2016 On ... session at 4,833.32, down 0.22%; the Dow Jones Industrial Average ... 500 closed at 2,085.45, down 0.17%. Stock-Callers.com has initiated coverage ... INFI ), Nektar Therapeutics (NASDAQ: NKTR ), Aralez ... Inc. (NASDAQ: BIND ). Learn more about these ...
(Date:6/23/2016)... (PRWEB) , ... June 23, 2016 , ... ... on quality, regulatory and technical consulting, provides a free webinar on ... on July 13, 2016 at 12pm CT at no charge. , Incomplete investigations ...
(Date:6/23/2016)... -- Amgen (NASDAQ: AMGN ) today announced a ... sciences incubator to accelerate the development of new therapies ... QB3@953 was created to help high-potential life science and ... stage organizations - access to laboratory infrastructure. ... "Amgen Golden Ticket" awards, providing each winner with one ...
Breaking Biology Technology:
(Date:6/22/2016)... ANGELES , June 22, 2016 /PRNewswire/ ... identity management and verification solutions, has partnered ... edge software solutions for Visitor Management, Self-Service ... provides products that add functional enhancements ... partnership provides corporations and venues with an ...
(Date:6/15/2016)... , June 15, 2016 ... report titled "Gesture Recognition Market by Application Market - Global Industry ... - 2024". According to the report, the  global gesture ... in 2015 and is estimated to grow at ... billion by 2024.  Increasing application of ...
(Date:6/2/2016)... The Department of Transport Management (DOTM) ... million US Dollar project, for the , Supply ... Enrolment, and IT Infrastructure , to ... implementation of Identity Management Solutions. Numerous renowned international vendors participated ... was selected for the most compliant and innovative ...
Breaking Biology News(10 mins):