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:10/11/2017)... ... ... is a basic first aid supply for any work environment, but most personal eye wash ... if a dangerous substance enters both eyes? It’s one less decision, and likely quicker response ... piece. , “Whether its dirt and debris, or an acid or alkali, getting anything in ...
(Date:10/11/2017)... ... ... Disappearing forests and increased emissions are the main causes of the evolving air ... living in larger cities are affected by air pollution related diseases. , That is ... globally - decided to take action. , “I knew I had to take action ...
(Date:10/10/2017)... , ... October 10, 2017 , ... ... Center’s FirstHand program has won a US2020 STEM Mentoring Award. Representatives of the ... for Excellence in Volunteer Experience from US2020. , US2020’s mission is to change ...
(Date:10/10/2017)... ... October 10, 2017 , ... ... 13 prestigious awards honoring scientists who have made outstanding contributions ... scheduled symposium during Pittcon 2018, the world’s leading conference and exposition for laboratory ...
Breaking Biology Technology:
(Date:4/19/2017)... April 19, 2017 The global ... landscape is marked by the presence of several large ... held by five major players - 3M Cogent, NEC ... accounted for nearly 61% of the global military biometric ... in the global military biometrics market boast global presence, ...
(Date:4/17/2017)... -- NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or ... 2016 Annual Report on Form 10-K on Thursday April 13, 2017 ... ... Investor Relations section of the Company,s website at http://www.nxt-id.com  under ... http://www.sec.gov . 2016 Year Highlights: ...
(Date:4/13/2017)... April 13, 2017 According to a new market ... Identity Analytics, Identity Administration, and Authorization), Service, Authentication Type, Deployment Mode, Vertical, ... Market is expected to grow from USD 14.30 Billion in 2017 to ... of 17.3%. ... MarketsandMarkets Logo ...
Breaking Biology News(10 mins):