Navigation Links
T98G

Multiporator Transfection Protocol Protocol No. 4308 915.041 11/2000 Cell line T98G, human glioblastoma Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium IMDM / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Ariane Sling Martin-Luther-Universitt Halle Molekulare Neuroonkologie
Magedburger Str. 16 D-06097 Halle/Saale
Phone +49 345 5574050 Fax +49 345 5571412 e-mail: ariane.soeling@medizin.uni-halle.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 IMDM / 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 (10-20 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) 500 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 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 3-5 ml IMDM / 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: 77% Transfection rate: 39% based on the number of surviving cells Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Saccharomyces cerevisiae
2. Saccharomyces cerevisiae
3. Saccharomyces cerevisiae
4. Electrophoretic Karyotypes of Wine Strains of Saccharomyces cerevisiae
5. Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives in the yeast Saccharomyces cerevisiae: Detection in vivo using the Varian Cary Eclipse
6. Monitoring fluorescence resonance energy transfer (FRET) between GFP fusions in lysates of the yeast Saccharomyces cerevisiae using the Varian Cary Eclipse
Post Your Comments:
*Name:
*Comment:
*Email:


(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 , ... ... outstanding manufactures in 10 categories with over 30 nominees and well as the ... presented the new award and the event was hosted by CompanyWeek and Manufacturers ...
(Date:4/21/2017)... ... 21, 2017 , ... The University of Connecticut, in partnership ... three startups through the UConn Innovation Fund. The $1.5 million UConn Innovation Fund ... UConn. , The UConn Innovation Fund provides investments of up to $100,000 to ...
(Date:4/21/2017)... , ... April 21, 2017 , ... The AMA is ... high school graduates from across the nation. The scholarships are created through funds donated ... dues. , Scholarship criteria are set by the AMA Scholarship Committee, which is made ...
Breaking Biology Technology:
(Date:3/24/2017)... MILAN , March 24, 2017 The Controller ... Deputy Controller Mr. Abdulla Algeen have received the prestigious international ... Continue Reading ... ... small picture) and Deputy Controller Abdulla Algeen (small picture on the right) ...
(Date:3/23/2017)... 23, 2017 Research and Markets has announced ... & Trends - Industry Forecast to 2025" report to their ... The ... CAGR of around 8.8% over the next decade to reach approximately ... the market estimates and forecasts for all the given segments on ...
(Date:3/22/2017)... , March 21, 2017 Vigilant ... company serving law enforcement agencies, announced today the appointment ... as director of public safety business development. ... law enforcement experience, including a focus on the aviation ... his most recent position, Mr. Sheridan served as the ...
Breaking Biology News(10 mins):