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:3/24/2017)... YORK , March 24, 2017 ... ended the trading session at 5,817.69, down 0.07%; the ... at 20,656.58; and the S&P 500 closed at 2,345.96, ... as 4 sectors closed in green, 4 sectors finished ... day. This Friday, Stock-Callers.com has initiated reports coverage on ...
(Date:3/24/2017)... March 24, 2017 Agenus Inc. (NASDAQ: AGEN), ... antibodies and cancer vaccines, today announced participation at the ...  Annual William Blair and Maidstone Life Sciences conference "Cancer ... in New York, NY . Agenus ... 29 at 9:40 am: Robert B. Stein , ...
(Date:3/23/2017)... ... March 23, 2017 , ... Lajollacooks4u is proud to announce it has become the premiere ... cooking challenges for companies around the world, such as Illumina, HP and Qualcomm, and is ... for its increasing popularity is due to its new team building format, a way for ...
(Date:3/23/2017)... , March 23, 2017  Northwest Biotherapeutics ... DCVax® personalized immune therapies for solid tumor cancers, ... $7.5 million financing it announced last Friday, March ... to several institutional investors securities totaling 28,843,692 shares, ... share, and 10,000,000 shares of Class C Warrants ...
Breaking Biology Technology:
(Date:3/22/2017)... , March 21, 2017   Neurotechnology ... object recognition technologies, today announced the release of ... (SDK), which provides improved facial recognition using up ... on a single computer. The new version uses ... improve accuracy, and it utilizes a Graphing Processing ...
(Date:3/22/2017)... , March 21, 2017 Vigilant Solutions ... serving law enforcement agencies, announced today the appointment of ... director of public safety business development. Mr. ... enforcement experience, including a focus on the aviation transportation ... most recent position, Mr. Sheridan served as the Aviation ...
(Date:3/20/2017)... , March 20, 2017 PMD Healthcare announces ... spirometer and Wellness Management System (WMS), a remote, real-time ... in 2010, PMD Healthcare is a Medical Device, Digital ... mission dedicated to creating innovative solutions that empower people ... that intent focus, PMD developed the first ever personal ...
Breaking Biology News(10 mins):