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:4/26/2017)... ... April 26, 2017 , ... WonderWorks, Myrtle ... to showcase the future of deep space exploration and inspire space enthusiasts. The ... spacecraft and includes a guest appearance by former Shuttle Astronaut Don Thomas. , ...
(Date:4/26/2017)... Hong Kong (PRWEB) , ... April 26, 2017 ... ... be hosted in EMEA and North America this May ... from May 16-18 , Donald H. Taylor, Chairman of the Learning and Performance ...
(Date:4/25/2017)... ... 2017 , ... Franz Inc ., an early innovator ... market leader for Semantic Graph Database technology, today announced Allegro ... effective system for developing and deploying applications to solve the challenges developers face ...
(Date:4/25/2017)... ... April 25, 2017 , ... ... Metrology Partners.     , Covalent’s Analytical Services unit provides high-quality data to ... measured within 24 hours of receipt. There are no price premiums, and customers ...
Breaking Biology Technology:
(Date:3/28/2017)... -- The report "Video Surveillance Market by ... Devices), Software (Video Analytics, VMS), and Service (VSaaS, Installation ... 2022", published by MarketsandMarkets, the market was valued at ... reach USD 75.64 Billion by 2022, at a CAGR ... considered for the study is 2016 and the forecast ...
(Date:3/23/2017)... Research and Markets has announced the addition of the "Global ... 2025" report to their offering. ... The Global Vehicle Anti-Theft System Market is ... next decade to reach approximately $14.21 billion by 2025. ... all the given segments on global as well as regional levels ...
(Date:3/20/2017)... 20, 2017 PMD Healthcare announces the release ... Wellness Management System (WMS), a remote, real-time lung health ... PMD Healthcare is a Medical Device, Digital Health, and ... to creating innovative solutions that empower people to improve ... focus, PMD developed the first ever personal spirometer, Spiro ...
Breaking Biology News(10 mins):