Navigation Links
721.221

Multiporator Transfection Protocol Protocol No. 4308 915.044 11/2001 Cell line 721.221, EBV transfected B cell line, MHC class I A, B, C negative,
MHC class II positive 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 721.221 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: 86.18% Transfection rate: 13.28% 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:12/2/2016)... PUNE, India , December 2, 2016 ... Billion by 2021, growing at a CAGR of 7.3% during the ... segment while hospitals and diagnostic laboratories segment accounted for the largest ... ... Complete report on global immunohistochemistry (IHC) market spread across 225 ...
(Date:11/30/2016)... 2016 /PRNewswire/ - Portage Biotech Inc. ("Portage" or "the ... excited to announce the formation of EyGen, Ltd. ... ophthalmology assets through proof of concept. EyGen,s lead ... Portage Pharmaceuticals Limited and being developed for topical ... anterior segment diseases. This agent has the potential ...
(Date:11/30/2016)... SAN DIEGO and BEIJING ... Ltd., a leading commercial provider of genomic services and ... expertise, announced today that it has completed a USD ... China Merchants Bank Co., Ltd.,s CMB International Capital Management ... SDIC Innovation Investment Management Co., Ltd. ("SDIC Innovation") and ...
(Date:11/30/2016)... ... November 30, 2016 , ... Energetiq Technology, a world leader ... generation, ultra-bright, Laser-Driven Light Source, the EQ-77, at the MRS Fall Exhibit 2016. ... offers higher radiance and irradiance from a truly broadband white light source. The ...
Breaking Biology Technology:
(Date:12/2/2016)... , Dec. 1, 2016   SoftServe , ... BioLock , an electrocardiogram (ECG) biosensor analysis ... a key IoT asset. The smart system ensures ... vehicle,s steering wheel and mobile devices to easily ... As vehicle technology advances, so too ...
(Date:11/29/2016)... 2016   Neurotechnology , a provider ... technologies, today released FingerCell 3.0, a software ... that run on low-power, low-memory microcontrollers. FingerCell ... than 128KB of memory, enabling it to ... limited on-board resources, such as: mobile phones, ...
(Date:11/22/2016)... -- According to the new market research report "Biometric System ... Voice), Multi-Factor), Component (Hardware and Software), Function (Contact and Non-contact), Application, and ... expected to grow from USD 10.74 Billion in 2015 to reach USD ... 2022. Continue Reading ... ...
Breaking Biology News(10 mins):