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:2/10/2016)... /PRNewswire/ - BioAmber Inc. (NYSE: BIOA ), a ... Mitsui & Co. Ltd., its partner in the ... investing an additional CDN$25 million in the joint venture ... 30% to 40%.  Mitsui will also play a stronger ... Sarnia , providing dedicated resources alongside ...
(Date:2/10/2016)... York, New York (PRWEB) , ... ... ... Pharmaceuticals Inc. (NASDAQ: REGN) today announced that it has joined the Human ... and immunotherapies for infectious diseases and cancer. , The Human Vaccines ...
(Date:2/10/2016)... ... February 10, 2016 , ... ... International Society of Pharmaceutical Engineering (ISPE) Rocky Mountain Chapter 21st Annual Vendor Exhibition ... to fill more than 100 tables for its annual event, which will run ...
(Date:2/10/2016)... FL (PRWEB) , ... February 10, 2016 , ... ... in regenerative medicine, has announced a new agreement with Singapore-based Global Stem Cells ... physicians from the Philippines, Thailand and Singapore in the latest adipose and bone ...
Breaking Biology Technology:
(Date:2/2/2016)... Feb. 2, 2016  Based on its recent ... Sullivan recognizes US-based Intelligent Retinal Imaging Systems (IRIS) ... Award for New Product Innovation. IRIS, a prominent ... North America , is poised to set ... diabetic retinopathy market. The IRIS technology presents superior ...
(Date:1/28/2016)... JOSE, Calif., Jan. 28, 2016 Synaptics (NASDAQ: SYNA ... results for its second quarter ended December 31, 2015. ... second quarter of fiscal 2016 increased 2 percent compared to the ... second quarter of fiscal 2016 was $35.0 million, or $0.93 per ... Non-GAAP net income for the first quarter of fiscal 2016 grew ...
(Date:1/22/2016)... 22, 2016 ... of the  "Global Behavioral Biometric Market ... --> http://www.researchandmarkets.com/research/4lmf2s/global_behavioral ) has announced ... Biometric Market 2016-2020"  report to their ... Markets ( http://www.researchandmarkets.com/research/4lmf2s/global_behavioral ) has announced the ...
Breaking Biology News(10 mins):