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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 l of electrocompetent cells. Homogenize by gently mixing with pipette several times.

  2. Transfer mixture into a prechilled cuvette.
  3. Wipe moisture from the cuvette and insert the cuvette into the device.

  4. Electroporation:

  5. Mode Prokaryotes O Voltage (V) 1440 V Time constant (T) 5 ms
  6. Immediately add 400 l LB medium and transfer into a sterile tube. Incubate at room temperature for 1 hour.
  7. Plate cells on minimal selective AB plates; incubate 12-72 hours.

Expected results:

Transformation efficiency up to 1-3 x 108 transformants/g of DNA.



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