Navigation Links
Escherichia coli K12

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.514 04/2002 Microorganism Escherichia coli K12 Cell type Bacteria, gram negative Molecules injected Plasmid DNA (pUC 19) Growth medium LB medium Washing solution Sterile, ice-cold water; (10% glycerol) Electroporation solution Sterile, ice-cold water; (10% glycerol) Outgrowth medium SOC medium (without antibiotics) Cuvette 1 mm gap width 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
Adapted from: High Efficiency Transformation by Electroporation Short Protocols in Molecular Biology
Second Edition Green Publishing Association and John Wiley & Sons, New York 1-22 1-23 Making electrocompetent cells:

1. Inoculate 500 ml LB medium with 2.5 ml of a fresh overnight culture of E. coli DH10B. Grow at 37 C with shaking to an O.D.600 of 0.5 to 0.6. 2. Chill cells on ice for 15 minutes and transfer to a prechilled centrifuge bottle. Harvest by centrifugation (20 minutes, 5,000 x g, 2-4 C). Resuspend pellet in 5 ml ice-cold water. Keep the cells cold during the entire procedure. 3. Wash twice with the original culture volume of ice-cold water. Centrifuge as above. Resuspend pellet by swirling in remaining liquid. 4a. If using the cells immediately, place suspension in a prechilled tube and centrifuge (10 minutes, 5,000 x g, 2-4 C). Resuspend the cells in ice-cold water to a final concentration of approximately 2 x 1011 cells/ml. Aliquote 40-300 l cells into prechilled centrifuge tubes. 4b. If freezing the cells for later use, add 40 ml of ice-cold 10% glycerol, mix and centrifuge for 10 minutes, at 5,000 x g and 2-4 C. Resuspend the cells in ice-cold 10% glycerol to a final concentration of approximately, 2 x 1011 cells/ml. Aliquote 40-300 l cells into prechilled centrifuge tubes. quick freeze on dry ice and store at 80 C.

Electroporation of cells:

  1. Add 1 l DNA (10 pg in water) to tubes containing 40 l electrocompetent cells. Homogenize by gently mixing with
    pipette several times.
  2. Transfer mixture to a prechilled cuvette. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes O Voltage (V) 1,700 V Time constant (T) 5 ms
  4. Immediately add 1 ml SOC medium and transfer to a sterile culture tube with a pasteur pipette. Incubate 30-
    60 minutes with moderate shaking at 37 C.
  5. Plate on LB plates containing the appropriate selection chemical.
Expected Results: Transformation efficiency up to 3.5 x 109 transformants/g of DNA.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Escherichia coli C600
2. Escherichia coli DH10B
3. Escherichia coli DH5
4. Escherichia coli C600
5. Escherichia coli K12
6. Escherichia coli DH10B
7. Escherichia coli DH5
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Escherichia coli

(Date:12/19/2014)... 2014 Ed Bilsky, Ph.D., vice president ... for Excellence in the Neurosciences (CEN), and professor of ... selected as a member of the prestigious Dana Alliance ... from Maine to receive this honor. , The ... by the Dana Foundation. Its mission is to ...
(Date:12/19/2014)... 19, 2014 In contrast to traditional ... with various colors, Valoya uses proprietary LED technology to ... best possible uniformity which is critical in low proximity ... spectral accuracy throughout its lifetime which is up to ... , "The investment cost was clearly higher when ...
(Date:12/19/2014)... Mass. , Dec. 18, 2014  Decision Resources ... dental biomaterial market will grow moderately through 2023 ... procedures and growing awareness of dental biomaterials all spur ... markets such as China and ... will bring dental procedures within reach for a larger ...
(Date:12/19/2014)... 18, 2014 Relmada Therapeutics, Inc. (OTCQB: RLMD), ... of chronic pain, announced the results of its annual ... was a transformational year in Relmada,s history and it ... company," said Sergio Traversa , chief executive officer ... on key financial, human resource and clinical development objectives ...
Breaking Biology Technology:UNE's Ed Bilsky Named Member of Dana Alliance for Brain Initiatives 2UNE's Ed Bilsky Named Member of Dana Alliance for Brain Initiatives 3Asia Pacific Dental Biomaterial Market Will Grow Strongly Through 2023 2Asia Pacific Dental Biomaterial Market Will Grow Strongly Through 2023 3Relmada Therapeutics Announces Results of Annual Shareholders Meeting 2
... MUMBAI, India, September 9 , ... Domain-Specific Learning,and Consulting Solutions for Health Care , ... (TIS)-has,recently announced the launch of its first business vertical-focused ... formally,launched this initiative with the appointment of Candida Chandorkar ...
... , WILMINGTON, N.C., Sept. 9 Jim ... - Wilmington, one of several new Sanford Rose Associates executive ... as COO of McGill Environmental Systems and CFO of House ... operations for several rapidly growing agribusiness, food processing, and distribution ...
... ... international study designed to find the genetic cause of Chiari-like Malformation and syringomyelia in dogs. ... cerebellum, descends out of the skull and crowds the spinal cord, putting pressure on both ... ...
Cached Biology Technology:Tata Interactive Systems Initiates Vertical-Focused Learning Solutions for Health Care, Pharmaceutical, and Biotechnology 2Tata Interactive Systems Initiates Vertical-Focused Learning Solutions for Health Care, Pharmaceutical, and Biotechnology 3Tata Interactive Systems Initiates Vertical-Focused Learning Solutions for Health Care, Pharmaceutical, and Biotechnology 4Sanford Rose Associates Opens Wilmington, N.C. Office 2DNA Genotek's Oragene●ANIMAL DNA Collection Kit Plays Key Role in Determining the Genetic Cause of Chiari Malformation in Dogs 2DNA Genotek's Oragene●ANIMAL DNA Collection Kit Plays Key Role in Determining the Genetic Cause of Chiari Malformation in Dogs 3
(Date:12/11/2014)... 2014  Data Sciences International (DSI), the global ... new series of digital telemetry implants to meet ... series, part of the PhysioTel™ Digital platform, was ... physiologic data when incorporating functional endpoints into repeat-dose ... toxicology studies has evolved from short ECG strips ...
(Date:12/10/2014)... N.C. , Dec. 9, 2014  Wake Forest Baptist ... education building for its School of Medicine. Funding for ... larger capital campaign that will be publicly launched next ... be located in the former 60 series R.J. Reynolds ... Innovation Quarter. Construction will begin immediately with plans to ...
(Date:12/10/2014)... SUNNYVALE, California , December 9, 2014 /PRNewswire/ ... Integrate with ITN,s BCARD Platform ... secure, cloud-based enterprise meeting scheduling solutions for business-to-business ... International, the world leader in mobile near-field communication ... solutions.      (Logo: http://photos.prnewswire.com/prnh/20140612/691055) , ...
Breaking Biology News(10 mins):New telemetry implants expected to change how large animal toxicology studies are conducted 2Wake Forest Baptist to Build New Medical Education Facility In Wake Forest Innovation Quarter 2Wake Forest Baptist to Build New Medical Education Facility In Wake Forest Innovation Quarter 3Wake Forest Baptist to Build New Medical Education Facility In Wake Forest Innovation Quarter 4Jifflenow And ITN International Bring Cutting-Edge Badge Scanning Technology To B2B Events 2Jifflenow And ITN International Bring Cutting-Edge Badge Scanning Technology To B2B Events 3
... will host its first Innovation Forum ( http://www.innovation-institute.org/dcforum/ ... Church, Va., on 6-8 November. The event is ... technologies and trends, and help high-tech professionals improve ... leaders from industry, academia and government are among ...
... public health issue and economic problem has been ... the University Denis Diderot in Paris, and the ... chlorine-resistant parasites, known as Cryptosporidium, which cause a ... significant mortality in immunodeficient patients, become virtually inactive ...
... report from the Commission for Environmental Cooperation (CEC) reveals ... to the environment15 percent for the United States and ... group of industrial facilities that are the largest generators ... that the leading role of the largest waste-producing facilities ...
Cached Biology News:First IEEE-USA Innovation Forum coming to Washington area in November 2Ultraviolet light helps to secure water supply 2Toxic releases down from North American industry leaders, increasing from other facilities 2
Mouse monoclonal antibody raised against a partial recombinant SCARB2. NCBI Entrez Gene ID = SCARB2...
Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor) (Alpha-2-PI) (Alpha-2-AP). [Source:Uniprot/SWISSPROT;Acc:P08697] Antigen: Recombinant Protein Epitope Signature Tag (PrEST)....
Mouse monoclonal antibody raised against a partial recombinant EOMES. NCBI Entrez Gene ID = EOMES...
... Membrane Proteins ,Contains sufficient components to ... 2-D gel analysis , ,Includes:, , ... , (contains sufficient reagents for extracting ... weight of 10 mg each) Kit ...
Biology Products: