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.

Transfer mixture into a prechilled cuvette.

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


3. Electroporation:


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.