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Agrobacterium Tumefaciens

Agrobacterium tumefaciens
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.502 – 12/2001
Microorganism Agrobacterium tumefaciens
Cell type Bacteria, gram negative
Molecules injected Plasmid DNA (in water)
Growth medium TB medium
Washing solution Sterile, cold water
Electroporation solution 10% glycerol
Outgrowth medium LB medium
Cuvette 1 mm gap width
Reference Mersereau M., et al • 1990 • Gene 90 • 149-151

Making electrocompetent cells:

  1. Grow cells at 30 °C to a density of O.D.600: 1.5-2.0.

  2. Harvest by centrifugation and wash five to seven times in sterile, cold water.

  3. Resuspend the cells in 10% glycerol to a final concentration of 4-6 x 1011 cell/ml and store frozen at –70 °C.

  4. For electroporation frozen cells were thawed in ice and diluted with sterile water to a density of 1 x 1010 cells/ml prior to use.

Electroporation of cells:

  1. Add 2 ng plasmid DNA to 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times.

    2. 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) 1440 V
    Time constant (T) 5 ms

  4. Immediately add 400 µl LB medium and transfer into a sterile tube. Incubate at room temperature for 1 hour.
  5. Plate cells on minimal selective AB plates; incubate 12-72 hours.

Expected results:

Transformation efficiency up to 1-3 x 108 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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