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:4/25/2017)... ... April 25, 2017 , ... Covalent ... Covalent’s Analytical Services unit provides high-quality data to clients, both faster ... hours of receipt. There are no price premiums, and customers are welcome to ...
(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)... ... ... The AMA is happy to announce that $48,000 in scholarships will be ... are created through funds donated by model aviation organizations and individuals, AMA members, and ... Scholarship Committee, which is made up of model aviation pilots and enthusiasts. The committee ...
(Date:4/20/2017)... Ultrecht, Netherlands (PRWEB) , ... April 20, 2017 ... ... Biometrics Technology today announced their strategic partnership to offer a full spectrum ... identity authentication, a comprehensive suite of biometric products and the ground-breaking proactive cybersecurity ...
Breaking Biology Technology:
(Date:3/30/2017)... 30, 2017 The research team of The ... (3D) fingerprint identification by adopting ground breaking 3D fingerprint minutiae recovery ... of speed and accuracy for use in identification, crime investigation, immigration ... ... A research team ...
(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:3/22/2017)... 21, 2017 Optimove , provider ... retailers such as 1-800-Flowers and AdoreMe, today announced ... and Replenishment. Using Optimove,s machine learning algorithms, these ... and replenishment recommendations to their customers based not ... of customer intent drawn from a complex web ...
Breaking Biology News(10 mins):