From 2014.igem.org

Revision as of 17:02, 14 October 2014

Protocols / Preparation and Transformation of R. radiobacter Cells

Preparation of electric-competent R. radiobacter cells

Protocol:

Rhizobium radiobacter was cultivated in 50 ml YEP medium containing the individually required antibiotics. At the time, the suspension reached an optical density (OD600) of ~0.4, the bacteria cells were precipitated by centrifuging them for 5 min at 4500 x g and 4 °C. While the supernatant was decanted, the precipitate was dissolved in 25 ml ice-cold 10 % glycerin. After repeating the centrifugation a second time, the precipitated bacteria were dissolved in 5 ml of the same glycerin again. The obtained competent cells were portioned (50 µl per 1.5 ml reaction vessel) and cooled down by liquid nitrogen. Longterm storage took place at -80 °C.

Transformation of electric-competent R. radiobacter cells

Protocol:

Before the frozen R. radiobacter cells can be used for transformation by electric poration, one reaction vessel containing ~50 µl competent cells has to be defrozen. After the suspension is completely thawn, bacteria and 1 µl of the desired plasmid were mixed in the cooled cuvette. The equipment involved in the process of electric poration was cooled down if possible. Under the condition of 25 μF, 200 Ω and 2.5 kV, R. radiobacter cells were transformed finally. Once the process was finished, 500 µl of preheated SOC medium (28 °C ) had to be added to the cells subsequently. After placing the transformed cells on ice for 30 min, cells were allowed to recover by shaking (220 rpm) for 3 h at 28 °C. Selection was achieved by growing the cells on antibiotics containing, solid YEP medium for 2 d at 28 °C.