Navigation Links
U937

Multiporator Transfection Protocol Protocol No. 4308 915.037 11/1999 Cell line U937, human leukemic monoblasts Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Steven Grant Box 980230 Medical College of Virginia Richmond VA 23 298 USA
Phone +1 804 828 5168 Fax +1 804 225 3788 e-mail: stgrant@hsc.vcu.edu
  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 RPMI / 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 2 x 106 cells/ml.
  4. Add and m ix plasmid DNA (25 g/ml final concentration, in bidistilled H2O).
  5. Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 620 V Time constant (T) 60 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 15 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 3 ml RPMI / 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: 50% Transfection rate: 24% based on the initial number of cells used for the experiment 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:4/28/2016)... ... 28, 2016 , ... As part of an ongoing global ... expanding its LATAM network and logistics capabilities. Enhancements have been made to ... trial projects. , The expansion will provide unmatched clinical trial logistics services for ...
(Date:4/27/2016)... (PRWEB) , ... April 27, 2016 , ... Cambridge ... by semantic web technology, today announced that it has been named to The Silicon ... sciences, financial services and other markets, Cambridge Semantics serves the needs of end users ...
(Date:4/27/2016)... ... April 27, 2016 , ... The Pittcon Organizing Committee is pleased ... has been a volunteer member of Committee since 1987. Since then, he has served ... and treasurer and was chairman for both the program and exposition committees. In his ...
(Date:4/27/2016)... , April 27, 2016 ... NSK) (OTCPink: NSKQB) ( Frankfurt : ... ihre Pressemitteilung vom 13. August 2015 die Genehmigung ... Finanzen um zusätzliche 200.000.000 Einheiten auf 400.000.000 Einheiten ... zu bringen. Davon wurden 157.900.000 Einheiten mit dem ...
Breaking Biology Technology:
(Date:3/23/2016)... WAKEFIELD, Massachusetts , March 23, 2016 ... kombiniert im Interesse erhöhter Sicherheit Gesichts- und ... Xura, Inc. (NASDAQ: MESG ... heute bekannt, dass das Unternehmen mit SpeechPro ... insbesondere aus der Finanzdienstleistungsbranche, wird die Möglichkeit ...
(Date:3/18/2016)... -- --> --> Competitive ... Unmanned Vehicles, Physical infrastructure and Perimeter Surveillance & Detection Systems ... security market and the continuing migration crisis in the ... has led visiongain to publish this unique report, which ... defence & security companies in the border security market ...
(Date:3/14/2016)... NXTD ) ("NXT-ID" or the "Company"), ... the airing of a new series of commercials on Time ... 21 st .  The commercials will air on Bloomberg TV, ... the Street show. --> NXTD ) ("NXT-ID" or ... market, announces the airing of a new series of commercials ...
Breaking Biology News(10 mins):