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:5/23/2016)... CA (PRWEB) , ... May 23, 2016 , ... ... public interest organization focused on molecular nanotechnology, announced the winners for the 2015 ... pioneer physicist Richard Feynman, are given in two categories, one for experiment and ...
(Date:5/20/2016)... MD (PRWEB) , ... May 20, 2016 , ... The ... with Listeria, as reported by Food Safety News on May 12, 2016(1), demonstrates the ... to Ted Olsen, CEO of Baltimore-based biotech firm, PathSensors, Inc. , ...
(Date:5/19/2016)... 2016 Regen BioPharma, ... RGBPP) announced today initiation of a preclinical development ... based cancer immunotherapeutic product leveraging its NR2F6 immunological ... a generation of cord blood derived killer cells ...  The product in development will be a "universal ...
(Date:5/18/2016)... ... 2016 , ... STACS DNA Inc., the sample tracking software company, today announced ... kit processing to help them save time and reduce errors. , Sexual Assault kits ... processed and victims informed of results. Due to a previous lack of tools, many ...
Breaking Biology Technology:
(Date:3/10/2016)... March 10, 2016 --> ... research report "Identity and Access Management Market by Component ... and Governance), by Organization Size, by Deployment, by Vertical, ... by MarketsandMarkets, The market is estimated to grow from ... by 2020, at a Compound Annual Growth Rate (CAGR) ...
(Date:3/9/2016)... 9, 2016 Nigeria ... more than 23,000 public service employees either did not ... their salary unlawfully.    --> Nigeria ... that more than 23,000 public service employees either did ... receiving their salary unlawfully.    --> DERMALOG, ...
(Date:3/3/2016)... -- FlexTech, a SEMI Strategic Association Partner, awarded five FLEXI ... Leadership in Education, and, in a category new this ... year of the FLEXI Awards and the winners join ... past years . Judging was done on a set ... by a panel of non-affiliated, independent, industry experts. ...
Breaking Biology News(10 mins):