Navigation Links
721.82

Multiporator Transfection Protocol Protocol No. 4308 915.045 11/2001 Cell line 721.82, EBV transfected B cell line, MHC class I positive,
MHC class II DR, DQ negative Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Kirsten Falk and Dr. Olaf Rtzschke Cellular Immunology
Max Delbrck Center for Molecular Medicine Robert-Rssle-Strasse 10 D-13125 Berlin-Buch
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 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 minutes, 200 x g, at room temperature). Remove supernatant.
  3. Resuspend the cells in medium, set the cell number to 1 x 106 cells/ml and centrifuge them (for 5 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!

  4. Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and mix plasmid DNA (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) 200 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 15 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 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 by viable 721.82 cells can be detected after 24 hours with a flow cytometer by costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT. Result: Survival rate: 67.6% Transfection rate: 12.9% based on the number of surviving cells. Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

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


(Date:10/12/2017)... ... October 12, 2017 , ... ... States multicenter, prospective clinical study that demonstrates the accuracy of the FebriDx® ... identifying clinically significant acute bacterial and viral respiratory tract infections by testing ...
(Date:10/11/2017)... ... October 11, 2017 , ... The ... endogenous context, enabling overexpression experiments and avoiding the use of exogenous expression plasmids. ... is transformative for performing systematic gain-of-function studies. , This complement to loss-of-function ...
(Date:10/11/2017)... the Netherlands and LAGUNA HILLS, Calif. ... The Institute of Cancer Research, London ... use MMprofiler™ with SKY92, SkylineDx,s prognostic tool to risk-stratify patients ... trial known as MUK nine . The University of ... trial, which is partly funded by Myeloma UK, and ICR ...
(Date:10/10/2017)... Los Angeles, CA (PRWEB) , ... ... ... Pharmaceuticals, Inc., a development-stage cancer-focused pharmaceutical company advancing targeted antibody-drug conjugate (ADC) ... all uses of targeted HPLN (Hybrid Polymerized Liposomal Nanoparticle), a technology developed ...
Breaking Biology Technology:
(Date:4/11/2017)... April 11, 2017 No two people ... at the New York University Tandon School of ... have found that partial similarities between prints are ... in mobile phones and other electronic devices can ... The vulnerability lies in the fact that fingerprint-based ...
(Date:4/5/2017)... 2017 KEY FINDINGS The global ... a CAGR of 25.76% during the forecast period of ... factor for the growth of the stem cell market. ... MARKET INSIGHTS The global stem cell market is ... geography. The stem cell market of the product is ...
(Date:3/30/2017)... March 30, 2017 Trends, opportunities and forecast ... behavioral), by technology (fingerprint, AFIS, iris recognition, facial recognition, ... others), by end use industry (government and law enforcement, ... and banking, and others), and by region ( ... Asia Pacific , and the Rest ...
Breaking Biology News(10 mins):