Navigation Links
A 20

Multiporator Transfection Protocol Protocol No. 4308 915.034 11/1999 Cell line A 20, mouse lymphoma B-cell line Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Isoosmolar Electroporation Buffer (PI) Culture medium RPMI 1640 / 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 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 Isoosmolar Electroporation buffer. When doing so, set the cell concentration to 3.8 x 106 cells/ml.
  4. Add and mix plasmid DNA (25 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) 600 V Time constant (T) 50 s No. of pulses (n) 2
  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 / 0.5% FCS, and centrifuge it for 5-10 minutes, 200 x g, at room temperature. Remove supernatant.
  9. Resuspend the cells in 3 ml RPMI 1640 / 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: 250% Transfection rate: 75% 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. 721.221
Post Your Comments:
*Name:
*Comment:
*Email:
TAG:

(Date:12/22/2014)... (PRWEB) December 22, 2014 ... peer-reviewed publication of original research, reviews and editorials ... and clinical practice, today published a provocative article ... the development, progression and potential treatment of prostate ... endocrinology research and proposes the possibility that there ...
(Date:12/19/2014)... and Hershey, PA (PRWEB) December 19, 2014 ... BJCT), a leading developer and manufacturer of needle-free ... into an agreement with Immunomic Therapeutics, Inc. (“ITI”) ... needle-free injection device with its LAMP™ vaccine platform. ... option for an exclusive Worldwide license to the ...
(Date:12/19/2014)... EVANSTON, Ill. , Dec. 19, 2014 ... to develop novel drugs for diseases of the central ... Ph.D., president and chief executive officer, will present at ... Dr. Riedel,s presentation will take place at 3:00 p.m. ... St. Francis in San Francisco, Calif. ...
(Date:12/19/2014)... Decision Resources Group finds that the Latin ... percent compound annual growth rate through 2023. This is ... the increasing adoption of dental implants. Growing aesthetic consciousness ... spur demand for dental biomaterials because they can improve ... Other key findings from Decision Resources Group,s ...
Breaking Biology Technology:Prostate Cancer Experts Dissect the Role of Follicle-stimulating Hormone in the Development, Progression and Potential Treatment of Prostate Cancer 2Prostate Cancer Experts Dissect the Role of Follicle-stimulating Hormone in the Development, Progression and Potential Treatment of Prostate Cancer 3Prostate Cancer Experts Dissect the Role of Follicle-stimulating Hormone in the Development, Progression and Potential Treatment of Prostate Cancer 4Bioject and Immunomic Therapeutics Enter into an Agreement for License of Needle-Free Technology for LAMP-vax Vaccines 2Bioject and Immunomic Therapeutics Enter into an Agreement for License of Needle-Free Technology for LAMP-vax Vaccines 3Naurex to Present at 33rd Annual J.P. Morgan Healthcare Conference 2Latin American Dental Biomaterial Market Will Expand at a 12 Percent Annual Growth Rate Through 2023 2Latin American Dental Biomaterial Market Will Expand at a 12 Percent Annual Growth Rate Through 2023 3
... , , BETHESDA, Md., July ... and NWBS) today announces that it will seek shareholder approval for ... AIM market ("AIM") (the "De-listing"). The Company plans to continue its ... Board Market ("U.S. listing"), and plans to file an S1 registration ...
... , SAN DIEGO, July 23 Anadys Pharmaceuticals, Inc. ... quarter 2009 financial results on Thursday, July 30, 2009, after the U.S. ... hold a conference call and webcast on Thursday, July 30, 2009 at ... results and highlights and to provide an update on its development programs. ...
... , SUNNYVALE, Calif., July 23 ... for the treatment of chronic total occlusions (CTOs), today announced the ... Chris previously held the position of Executive VP, Sales and Marketing ... Devices including the last 2 years at FlowCardia. , ...
Cached Biology Technology:Northwest Biotherapeutics Announces Plans to De-list From AIM and Consolidate its Stock Market Listing in U.S. 2Northwest Biotherapeutics Announces Plans to De-list From AIM and Consolidate its Stock Market Listing in U.S. 3Anadys Pharmaceuticals to Report Second Quarter 2009 Financial Results 2FlowCardia, Inc. Appoints Chris Barys to Chief Operating Officer 2
(Date:12/5/2014)... 2014 Increased emphasis on product quality, ... testing and inspection technologies. The convergence of industries ... range of innovative test solutions. This trend is ... which is generally more inclined towards communication, media ... strategies of test equipment vendors will cater to ...
(Date:11/21/2014)... DALLAS , November 18, 2014 ... Security Systems Market by Systems (Video, RFID, Access Control, ... (SEZ, Factories, Hotels, Banks, Government), Component Service Geography - ... Security Systems Market is projected to be around $25 ... Billion by 2020, growing at a CAGR of 8.69%. ...
(Date:11/18/2014)...  The Secure Identity & Biometrics Association (SIBA) ... the formation of The Airport Entry and Exit ... and Biometric Entry and Exit Solutions Framework for ... from BORDERPOL, the international non-profit organization that works ... regarding border security, traveler and migration systems. ...
Breaking Biology News(10 mins):Emerging Technologies Enable Smarter Monitoring of Assets, Push Industrial Testing, Inspection and Monitoring Market 2Emerging Technologies Enable Smarter Monitoring of Assets, Push Industrial Testing, Inspection and Monitoring Market 3Emerging Technologies Enable Smarter Monitoring of Assets, Push Industrial Testing, Inspection and Monitoring Market 4Industrial Security Systems Market Worth $38 Billion by 2020 2Industrial Security Systems Market Worth $38 Billion by 2020 3Identity Management, Document Authentication and Biometric Companies Form "The Airport Entry and Exit Working Group", Releasing the Framework to Help Guide the Department of Homeland Security and Congress Towards Solutions 2Identity Management, Document Authentication and Biometric Companies Form "The Airport Entry and Exit Working Group", Releasing the Framework to Help Guide the Department of Homeland Security and Congress Towards Solutions 3
... release is available in French . ... action to better control the sale of chemicals that ... Lunn, Minister of Natural Resources, and the Honourable Stockwell ... regulations for these chemicals, known as explosives precursors, will ...
... American,Association of Airport Executives President Chip ... Traveler (RT) program with the opening of,RT ... Reagan,Washington National Airport., "The expansion of ... in,the nation,s capital will further highlight the ...
... — Robots are fluent in their native language of 1 ... imprecise nature of human language. While scientists are making slow, ... responds to speech, gestures and body language, a more straightforward ... homes sooner. A team of researchers led by Charlie ...
Cached Biology News:New regulations tighten controls on restricted chemicals 2AAAE Applauds Expansion of Registered Traveler Program to Nation's Capital 2AAAE Applauds Expansion of Registered Traveler Program to Nation's Capital 3Robot fetches objects with just a point and a click 2Robot fetches objects with just a point and a click 3Robot fetches objects with just a point and a click 4
Criterion staining and blotting trays are plastic trays designed for staining one or two Criterion gels or performing western blot detection. The trays have a working capacity of up to 500 ml. Suppli...
... a wide range of next-generation features into ... new standard for flexible laboratory solutions. It ... including pipetting, dilution, dispensing, and integrations ... powerful and flexible as it is efficient ...
... The Biomek FX P is the latest ... design for greater positional accuracy and increased robustness, ... any application. It can be configured with either ... deck capacity, the Biomek FX P sets ...
BD Falcon, microtest ELISA plate 96 well flat-bottom, clear. Polystyrene, nonsterile, no lid (25/sp, 100/ca) Packaging: 25 / pack, 100 / case ...
Biology Products: