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


(Date:2/11/2016)... 11, 2016 Non-profit Consortium Aims to ... to Support Research and Discovery --> ... an ambitious plan to sequence 100,000 individuals. It is intended ... at least 7 of North and East Asian countries. ... phase, the project will focus on creating phased reference genomes ...
(Date:2/11/2016)... NEW YORK , Feb. 11, 2016  Bioethics International, ... how medicines are researched, developed, marketed and made accessible to ... BMJ Open had named the publication of the ... for 2015. The publication is also featured as one of ... published in the last year that are most frequently read. ...
(Date:2/11/2016)... Feb. 11, 2016  Dovetail Genomics™ LLC today announced ... program for a planned metagenomic genome assembly service. ... metagenomic genome assembly method in a talk on Friday, ... & Technology conference in Orlando, Fla. ... complex datasets is difficult. Using its proprietary ...
(Date:2/11/2016)... ... February 11, 2016 , ... Global Stem Cells Group, ... Bankok,Thailand-based Global Stem Cells Network (GSCN) to distribute exosome injection and other biological ... Mexico, Costa Rica, Dominican Republic, Colombia, Argentina, Nicaragua, Panama, El Salvador, Venezuela, Peru, ...
Breaking Biology Technology:
(Date:2/10/2016)... India , February 10, 2016 /PRNewswire/ ... --> According to 2016 iris recognition ... identification iris recognition is more widely accepted ... available with both fingerprint and iris recognition ... the user to avoid purchasing two individual ...
(Date:2/4/2016)... , Feb. 4, 2016 The ... apparently one of the most popular hubs of ... MetaHIT and other huge studies of human microbiota, ... past few years, the microbiome space has literally ... biomedical research. This report focuses on biomedical ...
(Date:2/2/2016)... YORK , Feb. 2, 2016 /PRNewswire/ ... facilities are primarily focused on medical screening ... measure point-of-care parameters. Wearable devices that facilitate ... user,s freedom of movement are being bolstered ... for human biomedical signal acquisition coupled with ...
Breaking Biology News(10 mins):