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:1/19/2017)... ... January 19, 2017 , ... ... and development (R&D), today announced the launch of Data Science Services ... the rapidly evolving field of precision medicine. , Data Science Services ...
(Date:1/19/2017)... , Jan. 19, 2017 AquaBounty Technologies, Inc. ... enhancing productivity in aquaculture and a majority-owned subsidiary of ... it has completed the listing of its common shares ... subscription from Intrexon. "AquaBounty,s listing on NASDAQ ... broaden our exposure to the U.S. markets as we ...
(Date:1/19/2017)... ... January 19, 2017 , ... DaVita Clinical Research ... of drug and device development, and Prism Clinical Research , a leader ... announced Verified Clinical Trials (VCT) has been selected by both companies ...
(Date:1/19/2017)... BD (Becton, Dickinson and Company) (NYSE: BDX ... host a live webcast of its Annual Meeting of Shareholders on ... The webcast can be accessed from the BD corporate website at ... 31, 2017. ... About BD BD is a global medical technology company that ...
Breaking Biology Technology:
(Date:12/7/2016)... According to a new market research report "Emotion Detection ... Voice Recognition), Service, Application Area, End User, And Region - Global Forecast to ... 6.72 Billion in 2016 to USD 36.07 Billion by 2021, at a Compound ... ... MarketsandMarkets Logo ...
(Date:12/7/2016)...   Avanade is helping Williams Martini Racing, ... history, exploit biometric data in order to critically analyse ... competitive edge against their rivals after their impressive, record-breaking ... has worked with Williams during the 2016 season to ... rate, breathing rate, temperature and peak acceleration) for key ...
(Date:12/6/2016)... , Dec. 6, 2016  Zimmer Biomet Holdings, Inc. ... has priced an offering of €500.0 million principal amount of ... principal amount of its 2.425% senior unsecured notes due 2026. ... to occur on December 13, 2016, subject to the satisfaction of ... annual basis. The Company intends ...
Breaking Biology News(10 mins):