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:2/4/2016)... ... 2016 , ... Franz Inc. , an early innovator ... been recognized As “ Best in Semantic Web Technology - USA & Leader ... it’s our priority to showcase prominent professionals who are excelling in their industry ...
(Date:2/3/2016)... 2016  With the growing need for better ... underway, therapies such as monoclonal antibodies, recombinant protein ... of indications are in high demand. Conventionally expression ... and production of these therapeutics. However, due to ... costs, novel approaches and novel expression systems are ...
(Date:2/3/2016)... ... February 03, 2016 , ... ProMIS Neurosciences ... targets (epitopes) specific to misfolded, propagating strains of Amyloid beta involved in Alzheimer’s ... therapeutics for Alzheimer’s. , Following on from the first misfolded Amyloid beta target ...
(Date:2/3/2016)... ... February 03, 2016 , ... ... aid in the rapid development and ongoing quality control of molecular assays targeting ... is extremely high,” Dr. Gregory R. Chiklis, President and CEO of ZeptoMetrix, relayed ...
Breaking Biology Technology:
(Date:2/2/2016)... --Technology Enhancements Accelerate Growth of X-ray Imaging This ... computed radiography markets in Thailand , ... (TIM). It provides an in-depth analysis of ... regional market drivers and restraints. The study offers revenue ... attractiveness, both for digital and computed radiography. Market participants ...
(Date:2/1/2016)... BURNABY, Canada , February 1, 2016 ... new technological advancements to drive global touchfree intuitive gesture ...   --> Rising sales of consumer electronics ... intuitive gesture control market size ... of consumer electronics coupled with new technological advancements to ...
(Date:1/28/2016)... -- Synaptics (NASDAQ: SYNA ), a leading developer of human ... December 31, 2015. --> --> ... 2 percent compared to the comparable quarter last year to $470.5 ... $35.0 million, or $0.93 per diluted share. ... quarter of fiscal 2016 grew 9 percent over the prior year ...
Breaking Biology News(10 mins):