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Team:Hannover/Protocols/Rhizobium Electroporation
From 2014.igem.org
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| - | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols | + | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / Preparation and Transformation of <i>R. radiobacter</i> Cells</h1> |
<h2>Preparation of electro-competent <i>R. radiobacter</i> cells</h2> | <h2>Preparation of electro-competent <i>R. radiobacter</i> cells</h2> | ||
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<tr><td><h4>Material:</h4> | <tr><td><h4>Material:</h4> | ||
<tr><td>10 % glycerin</td><td></td></tr> | <tr><td>10 % glycerin</td><td></td></tr> | ||
| - | <tr><td>YEP medium</td><td>10 g/l Bacto tryptone,<br> 10 g/l yeast extract,<br> 5.0 g/l NaCl | + | <tr><td>YEP medium</td><td>10 g/l Bacto tryptone,<br> 10 g/l yeast extract,<br> 5.0 g/l NaCl; pH 7.0</td></tr> |
<tr><td>antibiotics</td><td></td></tr> | <tr><td>antibiotics</td><td></td></tr> | ||
</table><br></br> | </table><br></br> | ||
| - | <p>< | + | <p><h2>Protocol:</h2></p> |
| - | <p class="text"><i>Rhizobium radiobacter</i> was cultivated in 50 ml YEP medium containing the individually required antibiotics. At the time, the suspension reached an optical density ( | + | <p class="text"><i>Rhizobium radiobacter</i> was cultivated in 50 ml YEP medium containing the individually required antibiotics. At the time, the suspension reached an optical density (OD<sub>600</sub>) 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.</p> |
<h2>Transformation of electro-competent <i>R. radiobacter</i> cells</h2> | <h2>Transformation of electro-competent <i>R. radiobacter</i> cells</h2> | ||
<table colspan="2"><tr><td><h4>Material:</h4></td> | <table colspan="2"><tr><td><h4>Material:</h4></td> | ||
| - | <tr><td>YEP media </td><td>10 g/l Bacto tryptone,<br> 10 g/l yeast extract,<br> 5.0 g/l NaCl,<br> 15 g/l Bacto Agar | + | <tr><td>YEP media </td><td>10 g/l Bacto tryptone,<br> 10 g/l yeast extract,<br> 5.0 g/l NaCl,<br> 15 g/l Bacto Agar; pH 7.0</td></tr><tr><td></td><td></td></tr></tr><tr><td></td><td></td></tr><tr><td>SOC medium</td><td>937.5 μl SOB media,<br>12.5 μl 2 M MgCl<sub>2</sub>,<br>50.0 μl 20 % (w/v) glucose</td></tr><tr><td>antibiotics</td><td></td></tr><tr><td><tr><td>cold cuvettes for electric-poration</td><td></td></tr></table> |
<br><br> | <br><br> | ||
| - | <p>< | + | <p><h2>Protocol:</h2></p> |
| - | <p class="text">Before the frozen <i>R. radiobacter</i> 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, <i>R. radiobacter</i> cells were transformed finally. Once the process was finished, 500 µl of preheated SOC medium (28 °C ) had to be added to the cells <u>subsequently</u>. After placing the <span id='a1'</span> 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.</p> | + | <p class="text">Before the frozen <i>R. radiobacter</i> 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, <i>R. radiobacter</i> cells were transformed finally. Once the process was finished, 500 µl of preheated SOC medium (28 °C) had to be added to the cells <u>subsequently</u>. After placing the <span id='a1'</span> 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.</p> |
</div> | </div> | ||
Latest revision as of 23:36, 17 October 2014
Protocols / Preparation and Transformation of R. radiobacter Cells
Preparation of electro-competent R. radiobacter cells
Material: | |
| 10 % glycerin | |
| YEP medium | 10 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 medium | 937.5 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.
