Navigation Links
721.221

Multiporator Transfection Protocol Protocol No. 4308 915.050 11/2001 Cell line 721.221, EBV transfected B cell line Transfection with pcDNA3.1/V5-His-TOPO (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS (200 U/ml penicillin; 200 g/ml streptomycin, 2 mM glutamine) Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Britta Eiz-Vesper Medizinische Hochschule Hannover Institut fr Transfusionsmedizin
Feodor-Lynen-Str. 21 D-30625 Hannover
Phone +49 160-3680653 Fax +49 511-5322079 e-mail: eiz-vesper.britta@mh-hannover.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 300 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, 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 Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 2 x 107 cells/ml.
  4. Add and mix 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) 400 V Time constant (T) 50 s No. of pulses (n) 2
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 5 ml RPMI / 10% FCS, and cultivate them in a little culture flask.
Detection methods for transfection:
Selection and limiting dilution assay for stable transfection after 48 hours. Result: Survival rate: 85% Transfection rate: 40% based on the number of surviving cells. Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. 721.221
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/3/2016)... , ... May 03, 2016 , ... ... at Boston CEO 2016 on May 31st and June 1st at The Four ... for leading executives in the life sciences, offering exclusive access to key decision ...
(Date:5/3/2016)... ... May 03, 2016 , ... Kerafast Inc., developers of an ... across the globe, today announced the availability of a Zika virus antibody from ... treatment and prevention measures for the Zika virus, the virus’s geographical distribution continues ...
(Date:5/3/2016)... LONDON , May 3, 2016 ... Report Assessing Developers and Producers of Those Competitor Biologics  ... Guide to Companies, Activities and Prospects ,  ... drug companies? And what are their sales potentials? ... There you see results, trends, opportunities and revenue forecasting. ...
(Date:5/3/2016)... ... May 03, 2016 , ... According to world renowned ... definitive prostate cancer treatment, patients traditionally had two main treatment options: surgery or radiation. ... be made. , New technology has enabled doctors to administer higher doses of ...
Breaking Biology Technology:
(Date:3/21/2016)... WAKEFIELD, Massachusetts , March 22, 2016 ... and facial recognition with passcodes for superior security ... MESG ), a leading provider of secure digital communications ... pilot their biometric technology and offer enterprise customers, particularly ... provide secure facial recognition and voice authentication within a ...
(Date:3/17/2016)... 17, 2016 ABI Research, the leader ... global biometrics market will reach more than $30 ... from 2015. Consumer electronics, particularly smartphones, continue to ... anticipated to reach two billion shipments by 2021 ... Pavlakis , Research Analyst at ABI Research. "Surveillance ...
(Date:3/15/2016)... 15, 2016 Yissum Research Development Company ... company of the Hebrew University, announced today the formation ... technology of various human biological indicators. Neteera Technologies has ... from private investors. ... detection of electromagnetic emissions from sweat ducts, enables reliable ...
Breaking Biology News(10 mins):