Navigation Links
Escherichia coli DH10B

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.513 04/2002 Microorganism Escherichia coli DH10B 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) 1,660 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 4 x 109 transformants/g of DNA.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Escherichia coli C600
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:2/28/2020)... ... 2020 , ... Designed primarily for laboratory and industrial use, ... thus simplifying setup, streamlining integration, and optimizing workflow. Capable of recording FHD (1080p) ... new HS Series cameras to be released. The HS Series includes cameras with ...
(Date:2/21/2020)... ... 20, 2020 , ... The dietary supplement industry now has ... listed on an ingredient panel is actually in their product. As a ... adoption of CertainT, which is designed to protect the intellectual property (IP) of ...
(Date:2/19/2020)... ... 2020 , ... Intech , the leader in contract ... unified global brand identity, as it celebrates its 20th anniversary. , Fueled by ... an enthusiasm for designing and manufacturing state-of-the-art medical devices, Intech has grown leaps ...
(Date:2/11/2020)... BALTIMORE (PRWEB) , ... February 11, 2020 , ... ... biosolids and residuals solutions services in North America, today announced that the City ... long-term solution to its biosolids management needs. Commencing in April of 2020, Synagro ...
Breaking Biology Technology:
(Date:3/5/2020)... ... March 05, 2020 , ... ... technology . The AlphaSTEM Testâ„¢ is the first, and still only, available ... Other technologies that attempt to provide information about the number and quality of ...
(Date:3/4/2020)... ... 04, 2020 , ... VGXI, Inc., a uniquely specialized CDMO ... as a finalist in the category of Best Contract Manufacturing Organization for the ... of a DNA vaccine against the novel coronavirus, COVID-19, under a CEPI funded ...
(Date:3/3/2020)... ... , ... Board-certified oral and maxillofacial surgeon, Dr. Michael Noffze offers experienced ... experience, Dr. Noffze can successfully extract problem and impacted wisdom teeth in teens and ... to the oral and overall health of most patients as these extra teeth can ...
Breaking Biology News(10 mins):