Navigation Links
721.174

Multiporator Transfection Protocol Protocol No. 4308 915.043 11/2001 Cell line 721.174, EBV transfected B cell line, MHC class I positive, MHC class II negative Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Kirsten Falk and Dr. Olaf Rtzschke Cellular Immunology
Max Delbrck Center for Molecular Medicine Robert-Rssle-Strasse 10 D-13125 Berlin-Buch
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells and centrifuge them (for 5 minutes, 200 x g, at room temperature). Remove supernatant.
  3. Resuspend the cells in medium, set the cell number to 1 x 106 cells/ml and centrifuge them (for 5 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!

  4. Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and mix plasmid DNA (20 g/ml final concentration, in bidistilled H2O).
  5. Add and mix plasmid DNA (12.5 g/ml final concentration, in bidistilled H2O).
  6. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  7. Electroporation:

    Mode Eukaryotes Voltage (V) 200 V Time constant (T) 40 s No. of pulses (n) 1
  8. After the pulse, allow the cell suspension to stand in the cuvette for 15 minutes at room temperature.
  9. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 3 ml RPMI /10% FCS, and cultivate them in a 55 mm culture dish.

Detection methods for transfection:
The expression of the plasmid pEGFP-N1 by viable 721.174 cells can be detected after 24 hours with a flow cytometer by costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT. Result: Survival rate: 72.54% Transfection rate: 20.77% based on the number of surviving cells. Results were measured 24 hours after transfection.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Ustilago maydis
Post Your Comments:
*Name:
*Comment:
*Email:
TAG:

(Date:8/21/2014)... (PRWEB) August 21, 2014 ... Fertilizer Industry” is a professional and in-depth ... The report provides basic Seaweed Fertilizer information, ... industry chain structure as well as industry ... analysis, including domestic market as well as ...
(Date:8/21/2014)... His Majesty Willem-Alexander, King of the Netherlands to ... Majesty Willem-Alexander, King of the Netherlands, has announced that ... Advanced Biofuels’ Project LIBERTY cellulosic ethanol plant in Emmetsburg, ... take part in the Grand Opening Ceremony to perform ... and tour the plant. POET-DSM is a joint venture ...
(Date:8/21/2014)... N.Y. (PRWEB) August 21, 2014 The ... the first private college in the Mid-Hudson Region to ... will now begin accepting applications from qualified “high-technology” businesses ... “We are very pleased to have been selected for ... Governor Cuomo’s transformative initiative to stimulate economic development in ...
(Date:8/21/2014)... 2014 Mathematic studies at the Program for ... funded by the Jeffrey Epstein VI Foundation , have ... visually identified for elimination. These aggressive cells are the driver ... a topological map of what to look for when examining ... while tumor cells can be extracted from biopsy, it,s very ...
Breaking Biology Technology:Internationals Seaweed Fertilizer Industry 2014 Development Trend Analysis in New Research Study at DeepResearchReports.com 2Internationals Seaweed Fertilizer Industry 2014 Development Trend Analysis in New Research Study at DeepResearchReports.com 3His Majesty Willem-Alexander, King of the Netherlands, to Attend Project LIBERTY opening 2His Majesty Willem-Alexander, King of the Netherlands, to Attend Project LIBERTY opening 3The College of New Rochelle Becomes First Private College in Mid-Hudson Region to Earn START-UP NY Designation 2The College of New Rochelle Becomes First Private College in Mid-Hudson Region to Earn START-UP NY Designation 3Science Power Jeffrey Epstein and Harvard University Fund Project to Visually Stop Tumor Growth 2
... Systems, Inc (OTC: OMCM.OB) today announced that it ranked ... of 500 of the fastest growing technology, media, telecommunications, ... Rankings are based on the percentage of fiscal year ... 445 percent during this period. OmniComm,s CEO, ...
... Beating the flu is already tough, but it has become even ... so that two antiviral drugs don,t slow it down anymore. ... Florida State and Brigham Young move closer to understanding why not, ... how it changes. The two drugs, amantadine and rimantadine, are ...
... and existing in the limbo between a solid and a ... efficiency of chemical catalysts that can create better nanoparticles or ... transitional state, a catalyst can achieve its utmost potential with ... to a pair of Duke University researchers. A catalyst is ...
Cached Biology Technology:OmniComm Ranks Number 220 on Deloitte's 2010 Technology Fast 500 for Fastest Growing Companies in North America 2OmniComm Ranks Number 220 on Deloitte's 2010 Technology Fast 500 for Fastest Growing Companies in North America 3Putting a bull's-eye on the flu: Detailing influenza's structure for drug targeting 2Smaller is better in the viscous zone 2
(Date:8/21/2014)... American singles found that during sex with a ... On average, men experience orgasm 85.1 percent of ... difference. For women, however, orgasm occurrence is less ... of the time during sex with a familiar ... sexual orientation, with lesbian women experiencing orgasm more ...
(Date:8/21/2014)... 2014 /PRNewswire-iReach/ -- LiveScan Service Provider  Binary ... a partnership with Gabriel Health Institute, one of ... Certified Nursing Assistant preparatory schools. The agreement ... member of Binary,s LiveScan service provider,s owner/operator team; ... its service capabilities by widening the service ...
(Date:8/21/2014)... window? It consists of radiations in the 3.3 to ... water in biological tissues. New theoretical findings show that ... water window. These could be the basis of an ... biological samples or to be used in high-precision spectroscopy. ... needed to efficiently generate the harmonic radiationswhich are multiples ...
Breaking Biology News(10 mins):Orgasm rates for single women less predictable than men's, vary by sexual orientation 2Orgasm rates for single women less predictable than men's, vary by sexual orientation 3Orgasm rates for single women less predictable than men's, vary by sexual orientation 4LiveScan Provider Binary Biometrics Announces Partnership with Preeminent CNA School, Gabriel Health Institute to Expand Services in Orlando Region 2LiveScan Provider Binary Biometrics Announces Partnership with Preeminent CNA School, Gabriel Health Institute to Expand Services in Orlando Region 3Water window imaging opportunity 2
... dynamic world of the developing brain, neural stem cells give ... newborn neurons then migrate along the stem cell fibers up ... scientists have discovered a key mechanism of this migration ... developmental processes and diseases, including cancer. The finding, ...
... the much larger marsupial thylacine by being better built ... large prey, say Australian scientists. Despite being armed ... larger energy needs than the dingo, the thylacine was ... stronger head and neck anatomy allowed it to subdue ...
... BOSTON (Sept. 5, 2007) Steady increases in ... decades, as well as rates of Type 2 ... Mayer USDA Human Nutrition Research Center on Aging ... explore the relationship between sugar-sweetened beverage consumption and ...
Cached Biology News:Neural stem cell study reveals mechanism that may play role in cancer 2Neural stem cell study reveals mechanism that may play role in cancer 3Neural stem cell study reveals mechanism that may play role in cancer 4Tassie tiger no match for dingo 2Sugary drinks, not fruit juice, may be linked to insulin 2Sugary drinks, not fruit juice, may be linked to insulin 3