Navigation Links
t-HUVEC

Multiporator Transfection Protocol Protocol No. 4308 915.049 11/2001 Cell line t-HUVEC, transformed human umbilical endothelial cells, EC304 (ATCC) Transfection with pcDNA3-EGFP (endotoxin-free DNA-preparation; in bidistilled H2O) Electroporation buffer Eppendorf Electroporation Buffer with 200 mOsmol/kg Culture medium RPMI 1640 + 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Ina Radtke, Edith Jasny Ruhr-Universitt Bochum Fakultt fr Chemie
Molekulare Neurobiochemie Universittsstrae 150 D-44780 Bochum
Phone +49 234 32 22067 Fax +49 234 32 14105 e-mail ina.radtke@ruhr-uni-bochum.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 minutes, 160 x g, at room temperature).
  2. Resuspend the cells in RPMI 1640 with 1% FCS, determine the number of cells and centrifuge them (for 5 minutes, 160 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. < li>Resuspend the cells in Electroporation Buffer with 200 mOsmol/kg (= 42% Eppendorf Hypoosmolar Electroporation Buffer + 58% Eppendorf Isoosmolar Electroporation Buffer). When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. 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) 670 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 10 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 3 ml RPMI / 10% FCS, and cultivate it in a 55 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pcDNA3-EGFP can be detected clearly after 24-48 hours under a fluorescence microscope. Result: Survival rate: > 95% Transfection rate: 70% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection .


'"/>

Source:


Page: All 1 2 3

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:2/11/2016)... 11, 2016   BioInformant announces the February ... Research Products, Opportunities, Tools, and Technologies – Market Size, ... The first and ... cell industry, BioInformant has more than a decade of ... market, by stem cell type. This powerful 175 page ...
(Date:2/11/2016)... ... February 11, 2016 , ... ... a new agreement with Bankok,Thailand-based Global Stem Cells Network (GSCN) to distribute exosome ... Latin American countries, including Mexico, Costa Rica, Dominican Republic, Colombia, Argentina, Nicaragua, Panama, ...
(Date:2/10/2016)... MONTREAL , Febr. 10, 2016 /PRNewswire/ - BioAmber Inc. ... is pleased to announce that Mitsui & Co. Ltd., ... bio-based succinic acid plant, is investing an additional CDN$25 ... equity, increasing its stake from 30% to 40%.  Mitsui ... of bio-succinic acid produced in Sarnia ...
(Date:2/10/2016)... Feb. 10, 2016  The Maryland House of Delegates ... announced that University of Maryland School of Medicine Dean ... University of Maryland Medical System President and CEO ... Medallion," the highest honor given to the public by ... Dean Reece and Mr. Chrencik for their ...
Breaking Biology Technology:
(Date:2/1/2016)...  Today, the first day of American Heart Month, ... a first of its kind workplace health solution that ... the first application of Watson ... and Welltok will create a new offering that combines ... delivered on Welltok,s health optimization platform. The effort is ...
(Date:1/25/2016)... -- Glencoe Software, the world-leading supplier of image data management ... the data management solution OMERO Plus for the newly ... Photo - http://photos.prnewswire.com/prnh/20160125/325328LOGO ... Phenotypic analysis measures the characteristics and behavior of cells, ... such as health and disease, the presence or absence ...
(Date:1/20/2016)... Minn. , Jan. 20, 2016   MedNet ... supports the entire spectrum of clinical research, is pleased ... 2015. MedNet,s significant achievements are the result of the ... iMedNet eClinical , it,s comprehensive, easy-to-use ... --> --> Key MedNet growth achievements ...
Breaking Biology News(10 mins):