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


(Date:12/9/2016)... KONG , Dec. 9, 2016 China Cord ... "Company"), China,s leading provider of cord ... cell storage services, today announced the results of its 2016 ... in Hong Kong S.A.R., China . ... ratified the re-appointment of KPMG Huazhen LLP as ...
(Date:12/8/2016)... , Dec. 8, 2016  The Board of ... (PLSG), Western Pennsylvania,s only pure ... leadership. In accordance with the succession plan developed by ... years, effective today, James (Jim) F. Jordan ... Officer, succeeding John W. Manzetti , who is ...
(Date:12/8/2016)... -- OncoSec Medical Incorporated ("OncoSec") (NASDAQ: ONCS ... today announced financial results for the fiscal first ... on our commitment to address an unmet medical ... pleased with the early clinical response data presented ... focused on advancing our lead program – ImmunoPulse® ...
(Date:12/8/2016)... SAN DIEGO , Dec. 8, 2016 /PRNewswire/ ... presented demonstrating the role of the Breast Cancer ... stage, ER+ breast cancer are most at-risk for ... therapy. Data include results from three studies advancing ... provide information related to tumor biology and inform ...
Breaking Biology Technology:
(Date:11/14/2016)... CLARA, Calif. , Nov. 14, 2016 ... the biometric identification market, Frost & Sullivan ... Frost & Sullivan Award for Visionary Innovation ... player in the biometric identification market by ... multi-modal verification solution for instant, seamless, and ...
(Date:6/22/2016)... American College of Medical Genetics and Genomics was once again ... of the fastest-growing trade shows during the Fastest 50 Awards ... Las Vegas . Winners are ... of the following categories: net square feet of paid exhibit ... 2015 ACMG Annual Meeting was ranked 23 out of 50 ...
(Date:6/22/2016)... , June 22, 2016 On Monday, ... call to industry to share solutions for the Biometric ... U.S. Customs and Border Protection (CBP), explains that CBP ... are departing the United States , ... and to defeat imposters. Logo - ...
Breaking Biology News(10 mins):