Navigation Links

Multiporator Transfection Protocol Protocol No. 4308 915.040 11/2000 Cell line U-266, human myeloma Transfection with Plasmid pEGFP-C1 (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 Frau Pfeifer Universittsklinikum Leipzig Molekulare Immunologie Johannisallee 30
D-04103 Leipzig Phone: +49 341 9725494 Fax: +49 341 9725489
  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 l/ml final concentration, in bidistilled H2O) The cell suspension must be free of air bubbles.
  5. Electroporation:

    Mode Eukaryotes Voltage (V) 600 V Time constant (T) 60 s No. of pulses (n) 1
  6. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  7. Carefully transfer the cell suspension from the cuvette into 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-C1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 62% Transfection rate: 23% based on the number of surviving cells. Results were measured 48 hours after transfection.



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:

(Date:6/27/2016)... , June 27, 2016  Liquid Biotech ... announced the funding of a Sponsored Research Agreement ... circulating tumor cells (CTCs) from cancer patients.  The ... in CTC levels correlate with clinical outcomes in ... These data will then be employed to support ...
(Date:6/24/2016)... ... June 24, 2016 , ... While the majority of ... the Cary 5000 and the 6000i models are higher end machines that use the ... of the spectrophotometer’s light beam from the bottom of the cuvette holder. , ...
(Date:6/23/2016)...   Boston Biomedical , an industry leader ... target cancer stemness pathways, announced that its lead ... Designation from the U.S. Food and Drug Administration ... gastroesophageal junction (GEJ) cancer. Napabucasin is an orally ... stemness pathways by targeting STAT3, and is currently ...
(Date:6/23/2016)... ... June 23, 2016 , ... Charm Sciences, Inc. is pleased ... received AOAC Research Institute approval 061601. , “This is another AOAC-RI approval of ... Salter, Vice President of Regulatory and Industrial Affairs. “The Peel Plate methods perform ...
Breaking Biology Technology:
(Date:5/20/2016)... -- VoiceIt is excited to announce its new marketing ... working together, VoiceIt and VoicePass will offer an ... slightly different approaches to voice biometrics, collaboration between ... Both companies ... "This marketing and technology partnership allows VoiceIt ...
(Date:5/12/2016)... -- , a brand of Troubadour Research ... the Q1 wave of its quarterly wearables survey. A ... to a program where they would receive discounts for ... "We were surprised to see that so ... , CEO of Troubadour Research, "primarily because there are ...
(Date:5/3/2016)... 3, 2016  Neurotechnology, a provider of high-precision ... Automated Biometric Identification System (ABIS) , a complete ... MegaMatcher ABIS can process multiple complex biometric transactions ... of fingerprint, face or iris biometrics. It leverages ... and MegaMatcher Accelerator , which have been ...
Breaking Biology News(10 mins):