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Team:Hannover/Protocols/Transformation/Infiltration
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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> / Infiltration of <i>N. tabacum</i></h1> |
<span id='a2'></span><table colspan="2" width="450px"><tr><td><h4>Material:</h4></td> | <span id='a2'></span><table colspan="2" width="450px"><tr><td><h4>Material:</h4></td> | ||
<tr><td>Infiltrations medium</td><td>50 mM MES,<br> 2 mM Na<sub>2</sub>HPO<sub>4</sub>; pH: 5.5,<br> 28 mM α-Glucose,<br>100 μM Acetosyringon</td></tr><tr><td>syringes</td><td></td><tr><td>plastic bags</td><td></td></table> | <tr><td>Infiltrations medium</td><td>50 mM MES,<br> 2 mM Na<sub>2</sub>HPO<sub>4</sub>; pH: 5.5,<br> 28 mM α-Glucose,<br>100 μM Acetosyringon</td></tr><tr><td>syringes</td><td></td><tr><td>plastic bags</td><td></td></table> | ||
Latest revision as of 23:37, 17 October 2014
Protocols / Infiltration of N. tabacum
Material: |
|
| Infiltrations medium | 50 mM MES, 2 mM Na2HPO4; pH: 5.5, 28 mM α-Glucose, 100 μM Acetosyringon |
| syringes | |
| plastic bags |
Protocol:
To reach an optical density (OD600) of ~0.6 to 0.9, R. radiobacter cells were cultivated at 28 °C. After centrifuging the cells for 15 min at 3000 x g and 4 °C, the precipitate was kept and dissolved in infiltration medium. In contrast to the first dissolving agent, the infiltration medium for the second preparation step contained acetosyringon additionally. For the infiltration of Nicotiana tabacum leaves, syringes were loaded with 2 ml bacteria suspension. With low but constant pressure, R. radiobacter cells were injected into the leaf tissue. Treated plants were covered for 2 d. If the plants were not directly available, the prepared bacteria cells were stored at 4 °C temporarily.
Widely open stoma increase the transformation success. Therefore cultivate the plants at high humidity and water the plants before the treatment. It is was also experienced that completely differentiated leaves gave the best results.
Safety first! Protect your face with a mask!
