Navigation Links
293 T

Multiporator Transfection Protocol Protocol No. 4308 915.029 11/1999 Cell line 293 T, human embryonal kidney Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 10% FCS Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Hans Hcker Institut fr Med. Mikrobiologie, Immunologie und Hygiene
Technische Universitt Mnchen Trogerstr. 4a D-81675 Mnchen
Phone +49 89 4140 4184 Fax +49 89 4140 4183 e-mail: hans.haecker@lrz.tu-muenchen.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 DMEM / 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 Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 2.5 x 106 cells/ml..
  4. Add and mix plasmid DNA (12.5 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) 300 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 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 3-5 ml DMEM / 10% FCS, and cultivate it in a 55 mm culture dish.

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: 55% Transfection rate: 18% based on the number of surviving cells
10% based on the initial number of cells used for the experiment

Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Ustilago maydis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/19/2017)... (PRWEB) , ... June 19, 2017 , ... ... solutions for clinical development reported today that it is launching two new additions ... company will be demonstrating new capabilities at the DIA 2017 Annual Meeting in ...
(Date:6/16/2017)... ... , ... CTNext , Connecticut’s go-to resource for entrepreneurial support, today announced ... Chelsea Piers in Stamford. , Nine finalists, all of whom are Connecticut-based companies and ... to secure $10,000 awards to help support business growth. The winners included:, ...
(Date:6/15/2017)... ... 2017 , ... DuPont Industrial Biosciences (DuPont) announced that Jan ... Bloomberg’s 2017 Sustainable Business Summit: Seattle this Thursday, June 15, at 4:10 ... panel titled “Developing a Corporate Renewables Strategy.” , “Consumers want to buy ...
(Date:6/14/2017)... ... ... Diagenode, a leading global provider for complete solutions for ... technology specific for ChIP-sequencing from the CeMM for low ... (ChIP-seq) allows the study of histone modifications and transcription ...
Breaking Biology Technology:
(Date:3/30/2017)... KONG , March 30, 2017 The ... a system for three-dimensional (3D) fingerprint identification by adopting ground breaking ... into a new realm of speed and accuracy for use in ... at an affordable cost. ... ...
(Date:3/24/2017)... , March 24, 2017 The Controller General of ... Mr. Abdulla Algeen have received the prestigious international IAIR Award ... Continue Reading ... ... and Deputy Controller Abdulla Algeen (small picture on the right) have received ...
(Date:3/22/2017)... YORK , March 21, 2017 ... Marketing Cloud used by retailers such as 1-800-Flowers ... its platform — Product Recommendations and Replenishment. Using Optimove,s ... give more personalized product and replenishment recommendations to ... but also on predictions of customer intent drawn ...
Breaking Biology News(10 mins):