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: gerhild.taimor@physiologie.med.uni-giessen.de
  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.


'"/>

Source:


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:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... , June 27, 2016  Sequenom, Inc. (NASDAQ: ... enabling healthier lives through the development of innovative products ... the United States denied its ... the claims of Sequenom,s U.S. Patent No. 6,258,540 (",540 ... criteria established by the Supreme Court,s Mayo Collaborative Services ...
(Date:6/27/2016)... PHILADELPHIA , June 27, 2016  Liquid ... today announced the funding of a Sponsored Research ... study circulating tumor cells (CTCs) from cancer patients.  ... changes in CTC levels correlate with clinical outcomes ... therapies. These data will then be employed to ...
(Date:6/24/2016)... ... ... Researchers at the Universita Politecnica delle Marche in Ancona combed medical journal articles ... findings are the subject of a new article on the Surviving Mesothelioma website. ... blood, lung fluid or tissue of mesothelioma patients that can help point doctors to ...
(Date:6/23/2016)... 2016 A person commits a crime, and the ... track the criminal down. An outbreak of foodborne ... Administration (FDA) uses DNA evidence to track down the bacteria ... far-fetched? It,s not. The FDA has increasingly used a complex, ... foodborne illnesses. Put as simply as possible, whole genome sequencing ...
Breaking Biology Technology:
(Date:5/12/2016)... WearablesResearch.com , a brand of Troubadour Research & ... Q1 wave of its quarterly wearables survey. A particular ... a program where they would receive discounts for sharing ... "We were surprised to see that so many ... CEO of Troubadour Research, "primarily because there are segments ...
(Date:4/28/2016)... FRANCISCO and BANGALORE, India , ... of EdgeVerve Systems, a product subsidiary of Infosys (NYSE: ... provider, today announced a global partnership that will ... way to use mobile banking and payment services. ... is a key innovation area for financial services, but it ...
(Date:4/26/2016)... -- Research and Markets has announced the ...  report to their offering.  , ,     (Logo: ... forecast the global multimodal biometrics market to grow ... 2016-2020.  Multimodal biometrics is being implemented ... healthcare, BFSI, transportation, automotive, and government for controlling ...
Breaking Biology News(10 mins):