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:5/4/2016)... ... May 05, 2016 , ... CereScan, the nation’s leader in ... during National Stroke Awareness Month in May. An infographic created by CereScan ... CereScan will donate $1 up to a maximum of $3,000 through users ...
(Date:5/4/2016)... 4, 2016 According to a ... Market - Global Industry Analysis, Size, Share, Growth, Trends, ... anticipated to expand at a CAGR of 17.1% from ... 2024. Metabolomics is the extensive study of ... tissues or organisms. Together, these small molecules and their ...
(Date:5/3/2016)... ... May 03, 2016 , ... In a list published by the Boston ... 76 fastest-growing private companies; a small percentage of the state's 615,000+ small businesses. The ... percent change in revenue from 2012 to 2015. , As this award ...
(Date:5/3/2016)... - And Other Rising Companies ... Those Competitor Biologics  - Biosimilar Drug Producers ... ,  Who are the most important and ... their sales potentials? Discover, in our updated survey, organisations, ... and revenue forecasting. Visiongain,s new ...
Breaking Biology Technology:
(Date:4/19/2016)... , UAE, April 20, 2016 ... be implemented as a compact web-based "all-in-one" system solution ... the biometric fingerprint reader or the door interface with ... of modern access control systems. The minimal dimensions of ... ID readers into the building installations offer considerable freedom ...
(Date:4/13/2016)...  IMPOWER physicians supporting Medicaid patients in ... standard in telehealth thanks to a new partnership with ... IMPOWER patients can routinely track key health measurements, such ... and, when they opt in, share them with IMPOWER ... local retail location at no cost. By leveraging this ...
(Date:3/23/2016)... 23, 2016 ... Gesichts- und Stimmerkennung mit Passwörtern     ... MESG ), ein führender Anbieter digitaler ... mit SpeechPro zusammenarbeitet, um erstmals dessen Biometrietechnologie ... die Möglichkeit angeboten, im Rahmen mobiler Apps ...
Breaking Biology News(10 mins):