Navigation Links
Escherichia coli K12

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.514 04/2002 Microorganism Escherichia coli K12 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 ic e 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 V oltage (V) 1,700 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 3.5 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 C600
5. Escherichia coli K12
6. Escherichia coli DH10B
7. Escherichia coli DH5
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:9/12/2018)... ROCHELLE, Va. (PRWEB) , ... September 12, 2018 ... ... that Lauren Bronich-Hall, a quality management professional and former Senior Director of Regulatory ... Expert Consultant. , As Senior Director of Regulatory Affairs and Quality Systems ...
(Date:9/7/2018)... Wis. (PRWEB) , ... September 06, 2018 , ... ... which runs from September 25th-28th is a meeting of “The industry’s preeminent event ... AptaFluor®, provide drug discovery scientists powerful tools to study these emerging targets in ...
(Date:9/7/2018)... ... September 06, 2018 , ... ... neuromuscular disease, has been validated to be as effective as manual pathology. The ... Staining in Frozen Muscle Biopsies,” was published in the Archives of Pathology & ...
(Date:9/1/2018)... (PRWEB) , ... August 31, 2018 , ... ... research results of the impact of a proprietary biofield energy treated nutraceutical supplement ... assessed biomarkers for immune function response and blood, as well as, organ-specific inflammation. ...
Breaking Biology Technology:
(Date:8/31/2018)... Wisc. (PRWEB) , ... August 30, 2018 , ... ... measure inhibitor residence times as a component of drug discovery programs. Longer-target engagement ... these residence times are low throughput, require expensive instrumentation, or require the alteration ...
(Date:8/29/2018)... Calif. (PRWEB) , ... August 28, 2018 , ... ... cluster and cloud infrastructure automation software, announced that Data In Science Technologies has ... The HudsonAlpha Institute for Biotechnology is a non-profit genomics and genetics research ...
(Date:8/29/2018)... ANGELES (PRWEB) , ... August ... ... collaborative study validates first non-invasive technology to identify specific chromosomal abnormalities using ... U.S.-based Ovation Fertility and Australia-based Life Whisperer suggests ...
Breaking Biology News(10 mins):