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:4/25/2017)... ... 25, 2017 , ... Franz Inc ., an early ... and market leader for Semantic Graph Database technology, today announced ... most effective system for developing and deploying applications to solve the challenges developers ...
(Date:4/25/2017)... ... April 25, 2017 , ... Dr. Robert G. ... , proudly announced today that acclaimed physiatrist Matthew Terzella, MD, has joined the ... 2017. , Dr. Terzella completed his residency in Physical Medicine and Rehabilitation at ...
(Date:4/24/2017)... ... April 24, 2017 , ... ... denaturation in a cellular milieu; however, the broad application of this cellular target ... platforms with sensitive quantitative readouts. Cell-based thermal stabilization assays are valuable methods for ...
(Date:4/21/2017)... ... April 21, 2017 , ... Having worked on the design of the ... to introduce it to top lab design architects from around the country at the ... of Industrial Design and Engineering Greg Casey will be at the show, where they ...
Breaking Biology Technology:
(Date:3/20/2017)... At this year,s CeBIT Chancellor Dr. Angela Merkel ... came to the DERMALOG stand together with the Japanese Prime Minster Shinzo ... At the largest German biometrics company the two government leaders could see ... as well as DERMALOGĀ“s multi-biometrics system.   Continue ... ...
(Date:3/16/2017)... 2017 CeBIT 2017 - Against identity fraud with DERMALOG solutions "Made ... ... Used combined in one project, multi-biometric solutions provide a crucial contribution against identity ... Used combined in one project, multi-biometric ... ...
(Date:3/9/2017)... MELBOURNE , Australia , March ... clinical study data at the prestigious World Lung Imaging ... Dr. Andreas Fouras , was invited to deliver ... and pulmonary medicine. This globally recognised event brings together ... and share the latest developments in lung imaging. ...
Breaking Biology News(10 mins):