Navigation Links
Primary cells, rat heart muscle

Multiporator Transfection Protocol Protocol No. 4308 915.035 12/1999 Cell line Primary cells: Adult heart muscle cells from rat, 6 days in culture Transfection with Plasmid pCMV--galactosidase (in bidistilled H2O) Electroporation buffer Eppendorf Isoosmolar Electroporation Buffer (PI) Culture medium M199 / HEPES (2 mM carnithin, 5 mM taurine, 10 M cytosine-arabinofur.)+ 20% FCS Outgrowth medium HEPES-buffer (140 mM NaCl, 3.6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 11 mM glucose, 100 U/ml penicillin, 100 g/ml streptomycin, 20 mM HEPES, pH 7.4) without phenol red + 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Gerhild Taimor Justus-Liebig-Universitt Physiologisches Institut Aulweg 129 D-35392 Gieen
Phone +49 641 9947243 Fax +49 641 9947239 mail:
  1. Harvest the cells by collagenase treatment (0.4 mg/ml, 30 min., 37 C) and centrifuge them (for 2 minutes, 300 x g, at room temperature).
  2. Resuspend the cells in M199 / 0.5% FCS, determine the number of cells and centrifuge them (for 2 minute s, 300 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 Isoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 104 - 105 cells/ml.
  4. Add and mix plasmid DNA (5-20 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) 100 V Time constant (T) 50 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 from the cuvette to 3-5 ml HEPES medium without phenol red / 20% FCS, and cultivate them in a 35 mm culture dish.

Detection methods for transfection:

The expression of the plasmid pCMV--galactosidase can be detected clearly after 24-48 hours with X-gal-staining under a microscope.



Page: All 1 2

Related biology technology :

1. Human Primary Preadipocytes and Differentiated Adipocytes
2. Primary cells, rat heart endothel
3. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
4. Deliver siRNAs Into Primary Cells
5. High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells
6. Electroporation of Primary Bone Marrow Cells
7. Efficient Primary Structure Elucidation of Disulfide-bridged Peptides
8. Embryonic stem cells, mouse
9. Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR
Post Your Comments:

(Date:10/11/2017)... BioMarketing, a leading provider of patient support solutions, has announced ... network, which will launch this week. The VMS CNEs will ... to enhance the patient care experience by delivering peer-to-peer education ... professionals to help women who have been diagnosed and are ... ...
(Date:10/10/2017)... CALIF. (PRWEB) , ... October 10, 2017 , ... San ... part of its corporate rebranding initiative announced today. The bold new look is ... reach, as the company moves into a significant growth period. , It will also ...
(Date:10/10/2017)... 2017 SomaGenics announced the receipt of a ... RealSeq®-SC (Single Cell), expected to be the first commercially ... microRNAs) from single cells using NGS methods. The NIH,s ... accelerate development of approaches to analyze the heterogeneity of ... techniques for measuring levels of mRNAs in individual cells ...
(Date:10/9/2017)... ... October 09, 2017 , ... At its national board meeting in North Carolina, ... Harvard University’s Departments of Physics and Astronomy, has been selected for membership in ... team for the 2015 Breakthrough Prize in Fundamental physics for the discovery of the ...
Breaking Biology Technology:
(Date:4/13/2017)... 2017 UBM,s Advanced Design and Manufacturing event ... emerging and evolving technology through its 3D Printing and ... alongside the expo portion of the event and feature ... focused on trending topics within 3D printing and smart ... event will take place June 13-15, 2017 at the Jacob ...
(Date:4/11/2017)... 2017 No two people are believed ... New York University Tandon School of Engineering and ... that partial similarities between prints are common enough ... phones and other electronic devices can be more ... lies in the fact that fingerprint-based authentication systems ...
(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 ...
Breaking Biology News(10 mins):