Navigation Links
H73C11

Multiporator Transfection Protocol Protocol No. 4308 915.024 11/2001 Cell line H73C11, mouse-human, heteromyeloma (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Electroporation Buffer with 150 mOsmol/kg Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 Temperature RT (20-25 C) Reference Prof. Ulrich Zimmermann Lehrstuhl fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508 Fax +49 931 888 4509
e-mail: zimmerma@biozentrum.uni-wuerzburg.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 RPMI 1640 / 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 150 mOsmol/kg Electroporation Buf fer (= 68% Eppendorf Hypoosmolar Electroporation Buffer + 32% Eppendorf Isoosmolar Electroporation buffer). When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid 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) 160 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 RPMI 1640 / 10% FCS, and cultivate it in a 60 mm culture dish.
    Note: After pulsing, the cells should be incubated for 2-3 h at 37 C before any centrifugation is performed, to ensure resealing of the membrane.
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: 250% Transfection rate: 20% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Primary cells, rat heart endothel
2. Primary cells, rat heart muscle
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/4/2016)... Feb. 4, 2016  Sangamo BioSciences, Inc. (NASDAQ: ... today that Edward Lanphier , Sangamo,s president and ... progress of Sangamo,s ZFP Therapeutic ® development programs ... 2:40 pm ET on Thursday, February 11, 2016, at ... Conference. The conference is being held in ...
(Date:2/4/2016)... N.J. , Feb. 4, 2016  CytoSorbents ... immunotherapy leader commercializing its flagship CytoSorb® blood filter ... surgery patients around the world, announced that CEO ... present at the Source Capital Group,s 2016 Disruptive ... update on the company.  Conference ...
(Date:2/4/2016)...  Spherix Incorporated (Nasdaq: SPEX ) -- an intellectual property ... intellectual property, today provided an update on the Company,s ... of Texas and announcing that ... Partes Re-examination ("IPR") proceedings that VTech and Uniden ... initiated on only certain claims of two of the ...
(Date:2/4/2016)... ... ... Many of the engineers at FireflySci, Inc. have been manufacturing quartz and ... other cuvette manufacturers is their supercharged customer service and their extensive database of glass ... flow of inside information, they have recently revamped their manufacturing techniques to reduce lead ...
Breaking Biology Technology:
(Date:1/28/2016)... Synaptics (NASDAQ: SYNA ), a leading developer of human interface ... 31, 2015. --> --> ... percent compared to the comparable quarter last year to $470.5 million. ... million, or $0.93 per diluted share. --> ... of fiscal 2016 grew 9 percent over the prior year period ...
(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 ...
(Date:1/20/2016)... Jan. 20, 2016   MedNet Solutions , an ... spectrum of clinical research, is pleased to announce the ... achievements are the result of the company,s laser focus ... eClinical , it,s comprehensive, easy-to-use and highly affordable ... --> Key MedNet growth achievements in 2015 include: ...
Breaking Biology News(10 mins):