Navigation Links
B16

Multiporator Transfection Protocol Protocol No. 4308 915.042 11/2001 Cell line B16, mouse melanoma cells Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 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 with 0.05% trypsin-EDTA for 1 min, stop by adding DMEM and centrifuge them (for 5 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in DMEM / 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 guar antee 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) 400 V Time constant (T) 100 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 DMEM /10% FCS, and cultivate them in a 55 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pEGFP-N1 by viable B16 cells can be detected after 24-48 hours with a flow cytometer by costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT. Result: Survival rate: 68.91% Transfection rate: 78.21% based on the number of surviving cells. Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Azotobacter vinelandii
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:12/2/2016)... Dec. 2, 2016  The Multiple Myeloma Research Foundation ... MMRF CoMMpass Study SM —the largest and most comprehensive ... in multiple myeloma—will be presented at the 58 th ... Exposition in San Diego from ... treatment strategies, as well as identify pathways and targets ...
(Date:12/2/2016)... (PRWEB) , ... December 01, 2016 , ... ACEA Biosciences, ... its Phase I/II clinical trials for AC0010 at the World Conference on Lung Cancer ... providing an update on the phase I/II clinical trials for AC0010 in patients with ...
(Date:12/2/2016)... ... December 01, 2016 , ... The Conference Forum has ... three days and will take place on February 1-3, 2017 at the Roosevelt Hotel ... Gulley (NCI), the program provides a unique 360-degree approach, which addresses the most up-to-date ...
(Date:12/2/2016)... MA (PRWEB) , ... December 02, 2016 , ... Robots ... Light Event on December 3rd, 2016. The event, which is held on the ... work with helping Americans with Disabilities back into the workplace. Suitable Technologies is partnering ...
Breaking Biology Technology:
(Date:11/15/2016)... DUBLIN , Nov 15, 2016 Research ... - Global Forecast to 2021" report to their offering. ... ... reach USD 16.18 Billion by 2021 from USD 6.21 Billion in ... Growth of the bioinformatics market is driven by ...
(Date:11/14/2016)... CLARA, Calif. , Nov. 14, 2016 ... the biometric identification market, Frost & Sullivan ... Frost & Sullivan Award for Visionary Innovation ... player in the biometric identification market by ... multi-modal verification solution for instant, seamless, and ...
(Date:6/22/2016)... 22, 2016  The American College of Medical Genetics and ... Magazine as one of the fastest-growing trade shows during ... the Bellagio in Las Vegas . ... of growth in each of the following categories: net square ... number of attendees. The 2015 ACMG Annual Meeting was ranked ...
Breaking Biology News(10 mins):