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:10/12/2017)... ... October 12, 2017 , ... BioMedGPS announces expanded coverage of ... newest module, US Hemostats & Sealants. , SmartTRAK’s US Market for Hemostats and ... synthetic sealants and biologic sealants used in surgical applications. BioMedGPS estimates the market ...
(Date:10/12/2017)... CA (PRWEB) , ... October ... ... (https://www.onramp.bio/ ) has launched Rosalind™, the first-ever genomics analysis platform specifically designed ... bioinformatics complexity. Named in honor of pioneering researcher Rosalind Franklin, who made ...
(Date:10/11/2017)... ... 11, 2017 , ... The CRISPR-Cas9 system has ... and avoiding the use of exogenous expression plasmids. The simplicity of programming this ... gain-of-function studies. , This complement to loss-of-function studies, such as with RNAi ...
(Date:10/11/2017)... CA, USA (PRWEB) , ... October 11, 2017 , ... ... to take place on 7th and 8th June 2018 in San Francisco, CA. The ... influencers as well as several distinguished CEOs, board directors and government officials from around ...
Breaking Biology Technology:
(Date:4/6/2017)... April 6, 2017 Forecasts by ... Document Readers, by End-Use (Transportation & Logistics, Government & ... Gas & Fossil Generation Facility, Nuclear Power), Industrial, Retail, ... Are you looking for a definitive report ... ...
(Date:4/5/2017)... YORK , April 5, 2017 Today ... is announcing that the server component of the HYPR ... known for providing the end-to-end security architecture that empowers ... HYPR has already secured over 15 million ... makers including manufacturers of connected home product suites and ...
(Date:4/4/2017)... NEW YORK , April 4, 2017   ... solutions, today announced that the United States Patent and ... The patent broadly covers the linking of an iris ... the same transaction) and represents the company,s 45 th ... our latest patent is very timely given the multi-modal ...
Breaking Biology News(10 mins):