Navigation Links
HeLa

Multiporator Transfection Protocol Protocol No. 4308 915.030 12/1999 Cell line HeLa, human cervix epitheloid carcinoma Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Eppendorf AG Application Hotline D-22331 Hamburg
Phone +49 180 3 666 789 Fax +49 40 53990 125 e-mail: application-hotline@eppendorf.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 1 x 106 cells/ml.
  4. Add and mix pla smid DNA (10-20 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) 800 V Time constant (T) 100 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 them 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: 98% Transfection rate: 42% based on the number of surviving cells Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Kluyveromyces lactis
2. Lactococcus lactis
3. Lactococcus lactis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/21/2017)... ... 2017 , ... Frederick Innovative Technology Center, Inc. (FITCI), a ... technology start-ups, is hosting “Celebration Friday” (a festive gathering highlighting client success stories) ... and networking at 3:30 p.m. at FITCI’s 4539 Metropolitan Court location, off English ...
(Date:4/20/2017)... ... ... USDM Life Sciences , the leading risk management, technological innovation and ... announce Holger Braemer as Vice President of its Europe division and Managing ... , Braemer is an integral part of USDM’s expansion of services and solutions for ...
(Date:4/20/2017)... ... April 20, 2017 , ... Parallel6™ , the leader in ... today that they were named one of the 2017 Top 10 eClinical Trial ... in the pharmaceutical industry. , “We take pride in honoring Parallel6 as one of ...
(Date:4/19/2017)... ... 2017 , ... The Vibrating Orifice Aerosol Generator (VOAG) was ... droplets of known diameters for research applications such as for calibrating droplet measuring ... monodisperse droplets. , The VOAG requires forcing liquid out of an orifice ...
Breaking Biology Technology:
(Date:3/24/2017)... -- The Controller General of Immigration from Maldives Mr. ... have received the prestigious international IAIR Award for the most innovative high ... ... Maldives Immigration Controller ... (small picture on the right) have received the IAIR award for the ...
(Date:3/22/2017)... Lithuania , March 21, 2017   ... and object recognition technologies, today announced the release ... kit (SDK), which provides improved facial recognition using ... cameras on a single computer. The new version ... to improve accuracy, and it utilizes a Graphing ...
(Date:3/20/2017)... Pa. , March 20, 2017 PMD ... 2.0 personal spirometer and Wellness Management System (WMS), a ... Founded in 2010, PMD Healthcare is a Medical ... with a mission dedicated to creating innovative solutions that ... life. With that intent focus, PMD developed the first ...
Breaking Biology News(10 mins):