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Escherichia Coli DH10B

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 µl 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 µl cells into prechilled centrifuge tubes. quick freeze on dry ice and store at –80 °C.

Electroporation of cells:

  1. Add 1 µl DNA (10 pg in water) to tubes containing 40 µl 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/µg of DNA.

Eppendorf Contact Information

Eppendorf
In the United States:
Eppendorf North America, Inc.
102 Motor Parkway,
Hauppauge, NY 11788-5178

Outside the United States:
Eppendorf AG
Barkhausenweg 1
22339 Hamburg
Germany
Tel: ++ 49 40 53 801-0
Fax: ++ 49 40 53 801-556
Web Site: http://www.eppendorf.com


Customer Service: 800-645-3050

Fax Number: 516-334-7506

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