Team:Hannover/Protocols/Rhizobium Electroporation

From 2014.igem.org

Revision as of 23:36, 17 October 2014 by Anke (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Protocols / Preparation and Transformation of R. radiobacter Cells

Preparation of electro-competent R. radiobacter cells

Material:

10 % glycerin
YEP medium10 g/l Bacto tryptone,
10 g/l yeast extract,
5.0 g/l NaCl; pH 7.0
antibiotics


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 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 electro-competent R. radiobacter cells

Material:

YEP media 10 g/l Bacto tryptone,
10 g/l yeast extract,
5.0 g/l NaCl,
15 g/l Bacto Agar; pH 7.0
SOC medium937.5 μl SOB media,
12.5 μl 2 M MgCl2,
50.0 μl 20 % (w/v) glucose
antibiotics
cold cuvettes for electric-poration


Protocol:

Before the frozen R. radiobacter cells can be used for transformation by electroporation, 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 electroporation 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.

Gently rinsed with water and stained in EtOH overnight, electroporation cuvettes can be reused multiple times.