Navigation Links
Escherichia coli C600

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.511 04/2002 Microorganism Escherichia coli C600 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 C600. 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 trans fer 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) 1,900 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 2 x 109 transformants/g of DNA.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

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


(Date:2/11/2016)... WASHINGTON , Feb. 11, 2016   BioInformant ... strategic report, "Stem Cell Research Products, Opportunities, Tools, and ... 2020. ... specialize in the stem cell industry, BioInformant has more ... of the stem cell market, by stem cell type. ...
(Date:2/11/2016)... ... 2016 , ... Global Stem Cells Group, has ... The new facility will provide advanced protocols and state-of-the-art techniques in cellular medicine, ... , The new GSCG clinic is headed by four prominent Ecuadorian physicians, including ...
(Date:2/10/2016)... Md. , Feb. 10, 2016  The Maryland ... Busch , has announced that University of Maryland School ... PhD, MBA and University of Maryland Medical System President ... of the "Speaker,s Medallion," the highest honor given to ... of Delegates. Dean Reece and Mr. ...
(Date:2/10/2016)... and New York, New York (PRWEB) , ... ... ... Regeneron Pharmaceuticals Inc. (NASDAQ: REGN) today announced that it has joined ... new vaccines and immunotherapies for infectious diseases and cancer. , The ...
Breaking Biology Technology:
(Date:1/20/2016)... A market that just keeps on growing. ... explosion in genomics knowledge. Learn all about it in ... of dynamic trends are pushing market growth and company ... pharmacogenomics - pathogen evolution - next generation sequencing - ... understanding of the role of genetic material in Disease ...
(Date:1/20/2016)... SAN JOSE, Calif. , Jan. 20, 2016 ... leading developer of human interface solutions, today announced ... touch controller solution for wearables and small screen ... appliances such as printers. Supporting round and rectangular ... the S1423 offers excellent performance with moisture on ...
(Date:1/15/2016)... , Jan. 15, 2016 Recent publicized breaches ... to find new ways to ensure data security and ... iOS and Android that ties a ... transforming it into a hardware authorization token. Customer service ... their fingerprint on their KodeKey enabled device to verify ...
Breaking Biology News(10 mins):