Navigation Links
CHO

Multiporator Transfection Protocol Protocol No. 4308 915.031 12/1999 Cell line CHO, Chinese hamster ovary, Transfection with Rat-DPP IV (in bidistilled H2O), stable transfection Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium Alpha-Medium / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Sabine Grams Institut fr Molekularbiologie und Biochemie Arnimallee 22
D-14195 Berlin Phone +49 30 8445 1543 Fax +49 30 8445 1541
  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 Alpha-Medium / 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
    1 x 106< /sup> cells/ml.
  4. Add and mix plasmid DNA (5-10 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) 290 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 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 5 ml Alpha-Medium / 10% FCS, centrifuge it for
    5-10 minutes, 200 x g, at room temperature. Remove supernatant.
  9. Resuspend the cells in 2 ml Alpha-Medium / 10% FCS, and cultivate them in a 6 well microtiterplate.

Detection methods for transfection:

FACS method. Result: Survival rate: 98% (measured 48 hours after transfection) Transfection rate (stable): 50% based on the initial number of cells used for the experiment (after 10 days of incubation
in selection medium)


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Borrelia burgdorferi
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... 27, 2016  Global demand for enzymes is ... 2020 to $7.2 billion.  This market includes enzymes ... products, biofuel production, animal feed, and other markets) ... biocatalysts). Food and beverages will remain the largest ... consumption of products containing enzymes in developing regions.  ...
(Date:6/27/2016)...  Sequenom, Inc. (NASDAQ: SQNM ), a ... the development of innovative products and services, announced today ... States denied its petition to review decisions ... U.S. Patent No. 6,258,540 (",540 Patent") are not patent ... Supreme Court,s Mayo Collaborative Services v. Prometheus Laboratories decision.  ...
(Date:6/27/2016)... ... 27, 2016 , ... Parallel 6 , the leading software as a ... Reach Virtual Patient Encounter CONSULT module which enables both audio and video telemedicine ... team. , Using the CONSULT module, patients and physicians can schedule a face to ...
(Date:6/27/2016)... , ... June 27, 2016 , ... ... for Amgen, will join the faculty of the University of North Carolina ... professor of strategy and entrepreneurship at UNC Kenan-Flagler, with a focus on the ...
Breaking Biology Technology:
(Date:6/3/2016)... 2016 Das DOTM ... Nepal hat ein 44 Millionen ... Kennzeichen, einschließlich Personalisierung, Registrierung und IT-Infrastruktur, an ... und Implementierung von Identitätsmanagementlösungen. Zahlreiche renommierte internationale ... teilgenommen, aber Decatur wurde als konformste und ...
(Date:5/24/2016)... Calif. , May 24, 2016 Ampronix facilitates superior patient care by ... LMD3251MT  3D medical LCD display is the latest premium product recently added to the ... ... ... Sony 3d Imaging- LCD Medical Display- Ampronix News ...
(Date:5/3/2016)... , May 3, 2016  Neurotechnology, a ... the MegaMatcher Automated Biometric Identification System (ABIS) ... large-scale multi-biometric projects. MegaMatcher ABIS can process multiple ... using any combination of fingerprint, face or iris ... MegaMatcher SDK and MegaMatcher Accelerator , ...
Breaking Biology News(10 mins):