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:8/16/2017)... (PRWEB) , ... August 16, 2017 , ... ... beneficial microbe delivery system, announced it has secured $2M in funding from an ... City Angels, Carmen Innovations, and SVG Thrive Fund. With this investment, 3Bar is ...
(Date:8/15/2017)... spending the past two years building a state-of-the-art technology which consolidates ... this platform to healthcare stakeholders (hospitals, foundations, biopharma companies etc.) who ... vis a vis their members, under their own brand. Three recent ... ... ...
(Date:8/15/2017)... Boston, MA (PRWEB) , ... August 15, 2017 , ... ... unmet need that has compromised these disciplines for more than half a century. ... cannot be counted. It is widely known that molecular tags developed for this ...
(Date:8/15/2017)... , ... August 15, 2017 , ... ... shopping cart. Now mobile responsive, the new website makes it easy to navigate ... in between. Users can now find detailed product information, educational industry content and ...
Breaking Biology Technology:
(Date:5/16/2017)... -- Veratad Technologies, LLC ( www.veratad.com ), an innovative and ... solutions, announced today they will participate as a sponsor ... May 17, 2017, in Washington D.C.,s ... Identity impacts the lives of billions of ... digital world, defining identity is critical to nearly every ...
(Date:4/18/2017)... 18, 2017  Socionext Inc., a global expert in SoC-based imaging ... server, the M820, which features the company,s hybrid codec technology. A ... Tera Probe, Inc., will be showcased during the upcoming Medtec Japan ... at the Las Vegas Convention Center April ... Click here for ...
(Date:4/11/2017)... Research and Markets has announced the addition of the ... ... grow at a CAGR of 30.37% during the period 2017-2021. ... prepared based on an in-depth market analysis with inputs from industry ... over the coming years. The report also includes a discussion of ...
Breaking Biology News(10 mins):