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:8/21/2017)... Arbor, MI (PRWEB) , ... August 21, 2017 ... ... Innovation Awards: Healthcare Edition 2017. The awards recognize medical centers that have implemented ... by their efficiency of patient care protocols, competitive advantages, financial impact/value, and market ...
(Date:8/21/2017)... (PRWEB) , ... August 21, 2017 , ... ... firm is utilizing its extensive experience with Health Economics and Outcomes Research (HEOR) ... Health Record data. In 2014, US healthcare spending exceeded $3.0 trillion with nearly ...
(Date:8/21/2017)... Francisco, California (PRWEB) , ... August 21, 2017 , ... ... collaborate for a project, funded by a Bill and Melinda Gates Foundation ... contraception. , Bill & Melinda Gates Foundation, also known as the Gates Foundation, is ...
(Date:8/18/2017)... ... August 18, 2017 , ... OAI, a leading Silicon ... Microfluidics Industries, announces the new Model 800E front and backside, semi-automatic mask aligner ... mask aligners. OAI has already received and installed several orders for the ...
Breaking Biology Technology:
(Date:4/5/2017)... 2017 Today HYPR Corp. , leading ... component of the HYPR platform is officially FIDO® ... security architecture that empowers biometric authentication across Fortune 500 ... secured over 15 million users across the financial services ... home product suites and physical access represent a growing ...
(Date:4/4/2017)... , April 4, 2017   EyeLock LLC , ... that the United States Patent and Trademark Office (USPTO) ... covers the linking of an iris image with a ... and represents the company,s 45 th issued patent. ... is very timely given the multi-modal biometric capabilities that ...
(Date:3/30/2017)... -- On April 6-7, 2017, Sequencing.com will host the world,s ... at Microsoft,s headquarters in Redmond, Washington ... health and wellness apps that provide a unique, personalized ... is the first hackathon for personal genomics and the ... the genomics, tech and health industries are sending teams ...
Breaking Biology News(10 mins):