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Borrelia Burgdorferi

Borrelia burgdorferi
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.506 – 12/2001
Microorganism Borrelia burgdorferi B31
Cell type Bacteria, gram negative
Molecules injected Linear DNA (in water)
Growth medium Barbour-Stoenner-Kelly (BSK) II medium without gelatin
Washing solution PBS, without divalent cations; EPS (272 mM sucrose, 15% glycerol)
Electroporation solution EPS (272 mM sucrose, 15% glycerol)
Outgrowth medium BSK II medium
Cuvette 2 mm gap width
Reference Samuels D.S., et al • 1994 • Journal of Bacteriology 176 • 6045-6049

Making electrocompetent cells:

  1. Grow cells in 500 ml BSK II at 32 °C to a cell density of 3-7 x 107 cells/ml.

  2. Harvest by centrifugation (4,000 x g, 20 min., 4 °C). Wash twice in 60 ml cold PBS and pellet by centrifugation (3,000 x g, 10 min., 4 °C).

  3. Wash three times in 20 ml cold EPS and pellet by centrifugation (2,000 x g, 10 min., 4 °C).

  4. Resuspend the cells in 0.6 ml cold EPS.

Electroporation of cells:

  1. Add 0.3 to 1 µg DNA to 50 µl of electrocompetent cells on ice. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.

  2. Wipe moisture from the cuvette and insert the cuvette into the device.

  3. Electroporation:


    4. Mode Prokaryotes “O“
    Voltage (V) 2,500 V
    Time constant (T) 5 ms

  4. Immediately add 1 ml BSK II medium, transfer cells to a 15 ml tube and add another 9 ml of BSK II. Incubate for 24-48 hours at 32 °C with shaking.
  5. Plate 0.1 ml of culture on solid BSK II medium. Pellet the remaining 9.9 ml (4,000 x g, 10 min.), resuspend in 1 ml of supernatant and plate on selective plates. Incubate for 14 days, at 32 °C in a humidified 5% CO2 chamber.

Expected results:

Transformation efficiency up to 103 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

Web Site: http://www.eppendorfna.com
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