Navigation Links
H73C11

Multiporator Transfection Protocol Protocol No. 4308 915.024 11/2001 Cell line H73C11, mouse-human, heteromyeloma (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Electroporation Buffer with 150 mOsmol/kg Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 Temperature RT (20-25 C) Reference Prof. Ulrich Zimmermann Lehrstuhl fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508 Fax +49 931 888 4509
e-mail: zimmerma@biozentrum.uni-wuerzburg.de
  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 1640 / 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 150 mOsmol/kg Electroporation Buf fer (= 68% Eppendorf Hypoosmolar Electroporation Buffer + 32% Eppendorf Isoosmolar Electroporation buffer). When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid DNA (10-20 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) 160 V Time constant (T) 40 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 5 ml RPMI 1640 / 10% FCS, and cultivate it in a 60 mm culture dish.
    Note: After pulsing, the cells should be incubated for 2-3 h at 37 C before any centrifugation is performed, to ensure resealing of the membrane.
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: 250% Transfection rate: 20% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Primary cells, rat heart endothel
2. Primary cells, rat heart muscle
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... (PRWEB) , ... June 23, 2016 , ... ... on quality, regulatory and technical consulting, provides a free webinar on ... on July 13, 2016 at 12pm CT at no charge. , Incomplete investigations ...
(Date:6/23/2016)... 22, 2016  Amgen (NASDAQ: AMGN ) ... QB3@953 life sciences incubator to accelerate the development ... laboratory space at QB3@953 was created to help high-potential ... for many early stage organizations - access to laboratory ... Amgen launched two "Amgen Golden Ticket" awards, providing each ...
(Date:6/22/2016)... DIEGO , June 22, 2016 ... that will allow them to produce up to ... from one lot within one week. These high-quality, ... time laboriously preparing cells and spend more time ... possible through a proprietary, high-volume manufacturing process that ...
(Date:6/22/2016)... , June 22, 2016  According to Kalorama ... generation sequencing (NGS) market include significant efforts in ... sequencers.  More accessible and affordable sequencers, say the ... demand for consumables including sample prep materials.  The ... for Sample Preparation for Next Generation Sequencing (NGS) ...
Breaking Biology Technology:
(Date:6/2/2016)... June 2, 2016 Perimeter Surveillance ... Unmanned Systems, Physical Infrastructure, Support & Other Service  ... visiongain offers comprehensive analysis of the global ... will generate revenues of $17.98 billion in 2016. ... Inc, a leader in software and hardware technologies for ...
(Date:5/12/2016)... WearablesResearch.com , a brand of Troubadour ... from the Q1 wave of its quarterly wearables survey. ... receptivity to a program where they would receive discounts ... company. "We were surprised to see that ... LaColla , CEO of Troubadour Research, "primarily because there ...
(Date:4/26/2016)... India and LONDON ... Infosys Finacle, part of EdgeVerve Systems, a product ... and Onegini today announced a partnership to integrate ... solutions.      (Logo: http://photos.prnewswire.com/prnh/20151104/283829LOGO ... to provide their customers enhanced security to access ...
Breaking Biology News(10 mins):