Navigation Links
BALB/3T3

Multiporator Transfection Protocol Protocol No. 4308 915.023 11/1999 Cell line BALB/3T3, mouse, embryo 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 Eppendorf AG Application Hotline D-22331 Hamburg
Phone +49 180 3 666 789 Fax +49 40 53990 125 e-mail: application-hotline@eppendorf.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 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 5-10 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!

  3. Resuspend the cells in Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid DNA (5-20 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) 720 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 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 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 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 125% Transfection rate: 28% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Bifidobacterium animalis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:1/13/2017)... ... January 13, 2017 , ... FireflySci ... it has found among its diverse customer base. The latest entry in this ... brands electroporators including BTX and Bio-Rad. FireflySci is introducing three distinct varieties including ...
(Date:1/12/2017)... Carolina (PRWEB) , ... January 12, 2017 , ... Each ... the Eureka Index – a process that evaluates the patent estate of a company, ... Immunolight LLC , a biomedical firm leading the way in technologies that transform energy ...
(Date:1/12/2017)... ... January 12, 2017 , ... ... 26-year-old Lisa Rosendahl’s doctors gave her only a few months to live. Now ... combination that has stabilized Rosendahl’s disease and increased both the quantity and quality ...
(Date:1/12/2017)... , January 12, 2017 A new report published by Allied ... - Global Opportunity Analysis and Industry Forecast, 2014-2022," projects that the global in vitro ... growing at a CAGR of 15.07% during the forecast period. ... ... ...
Breaking Biology Technology:
(Date:12/15/2016)... , Dec. 15, 2016 Advancements ... experience, health wellness and wellbeing (HWW), and ... in three new passenger vehicles begin to ... gesture recognition, heart beat monitoring, brain wave ... facial monitoring, and pulse detection. These will ...
(Date:12/8/2016)... Dec. 8, 2016  Singulex, Inc., the leader in ... entered into a license and supply agreement with Thermo ... agreement provides Singulex access to Thermo Scientific BRAHMS PCT ... is used to diagnose systemic bacterial infection and ... to aid in assessing the risk of critically ...
(Date:12/6/2016)... , Dec. 6, 2016  Zimmer Biomet Holdings, Inc. (NYSE ... priced an offering of €500.0 million principal amount of its ... amount of its 2.425% senior unsecured notes due 2026. ... occur on December 13, 2016, subject to the satisfaction of customary ... basis. The Company intends to ...
Breaking Biology News(10 mins):