Preparation of Agrobacterium Electro-Competent Cells

Preparation of electro-competent cells of Agrobacterium tumefaciens

1.      Pick a single colony from a freshly grown plate of Agrobacterium and inoculate into 100 ml LB liquid medium in 250 ml autoclaved flask using sterile toothpick and incubate at 28 ⁰C for 48 hours with vigorous shaking.

2.      Add 5 ml of the 48 hours grown culture re-inoculate into 1 liter flask containing 250 ml of the same LB medium and incubate at 28 ⁰C untill OD₆₀₀ of cells become 0.5-1.0 (10¹⁰ cells/ml).

3.   Transfer the cells aseptically to ice cold 50 ml propylene tube and keep cool on ice for 30 minutes.

4.  Now centrifuge the cells at 4000 rpm for 10 minutes in a bench top refrigerated centrifuge machine at 4 ⁰C. Decant the supernatant and re-suspend the pelleted in 50 ml of sterile cold ddH₂O.

5. Again centrifuge the cells at 4000 rpm in the same centrifuge at 4 ⁰C for 10 minutes and decant the supernatant. Again re-suspend the pelleted cells in 25 ml of sterile cold ddH₂O.

6.      After another wash, re-suspend the cells in 10 ml sterile cold ddH₂O containing filter sterilized cold 10% glycerol. This wash should be repeated.

Finally re-suspend the the cells in 1-1.5 ml filter sterilized cold 10% glycerol, aliquote in 50 ml and store at -70 ^oC.