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:6/23/2016)... NEW YORK , June, 23, 2016  The ... students to envision new ways to harness living systems ... of Modern Art (MoMA) in New York ... more than 130 participating students, showcased projects at MoMA,s ... included Paola Antonelli , MoMA,s senior curator of ...
(Date:6/23/2016)... ... June 23, 2016 , ... Supplyframe, the Industry Network ... Supplyframe Design Lab . Located in Pasadena, Calif., the Design Lab’s mission is ... projects are designed, built and brought to market. , The Design Lab is ...
(Date:6/23/2016)... ... June 23, 2016 , ... In a new case report published ... how a patient who developed lymphedema after being treated for breast cancer benefitted from ... the paradigm for dealing with this debilitating, frequent side effect of cancer treatment. ...
(Date:6/23/2016)... Calif. , June 23, 2016  Blueprint Bio, ... biological discoveries to the medical community, has closed its ... Matthew Nunez . "We have received ... with the capital we need to meet our current ... essentially provide us the runway to complete validation on ...
Breaking Biology Technology:
(Date:5/3/2016)... VILNIUS, Lithuania , May 3, 2016 /PRNewswire/ ... today released the MegaMatcher Automated Biometric Identification ... deployment of large-scale multi-biometric projects. MegaMatcher ABIS can ... and accuracy using any combination of fingerprint, face ... of MegaMatcher SDK and MegaMatcher ...
(Date:4/26/2016)... , April 27, 2016 ... the  "Global Multi-modal Biometrics Market 2016-2020"  report to ... ) , The analysts forecast the ... CAGR of 15.49% during the period 2016-2020.  ... number of sectors such as the healthcare, BFSI, ...
(Date:4/14/2016)... , April 14, 2016 ... Malware Detection, today announced the appointment of Eyal ... new role. Goldwerger,s leadership appointment comes at ... heels of the deployment of its platform at several ... biometric technology, which discerns unique cognitive and physiological factors, ...
Breaking Biology News(10 mins):