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


(Date:10/12/2017)... ... , ... They call it the “hairy ball.” It’s an ... a system of linkages and connections so complex and dense that “it looks ... at Worcester Polytechnic Institute (WPI) and director of the university’s bioinformatics and computational ...
(Date:10/12/2017)... ... 2017 , ... BioMedGPS announces expanded coverage of SmartTRAK Business ... US Hemostats & Sealants. , SmartTRAK’s US Market for Hemostats and Sealants module ... and biologic sealants used in surgical applications. BioMedGPS estimates the market will grow ...
(Date:10/11/2017)... ... October 11, 2017 , ... The CRISPR-Cas9 ... enabling overexpression experiments and avoiding the use of exogenous expression plasmids. The simplicity ... for performing systematic gain-of-function studies. , This complement to loss-of-function studies, such ...
(Date:10/11/2017)...  VMS BioMarketing, a leading provider of patient support solutions, ... Educator (CNE) network, which will launch this week. The VMS ... care professionals to enhance the patient care experience by delivering ... health care professionals to help women who have been diagnosed ... ...
Breaking Biology Technology:
(Date:8/15/2017)... 15 2017   ivWatch LLC , a medical device company ... today announced receipt of its ISO 13485 Certification, the global standard ... Organization for Standardization (ISO®). ... Model 400 Continuous Monitoring device for the early detection of IV ... "This is an important milestone for ...
(Date:6/23/2017)... and ITHACA, N.Y. , ... and Cornell University, a leader in dairy research, today ... bioinformatics designed to help reduce the chances that the ... the onset of this dairy project, Cornell University has ... for Sequencing the Food Supply Chain, a food safety ...
(Date:5/16/2017)... 2017  Veratad Technologies, LLC ( www.veratad.com ), an ... identity verification solutions, announced today they will participate as ... 15 thru May 17, 2017, in Washington ... Center. Identity impacts the lives of ... quickly evolving digital world, defining identity is critical to ...
Breaking Biology News(10 mins):