Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB2"
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Golden Gate assembly of RibADET25 and RibADET48 in p15.01 and transformation of chemically competent NEB Turbo ''E.coli''. | Golden Gate assembly of RibADET25 and RibADET48 in p15.01 and transformation of chemically competent NEB Turbo ''E.coli''. | ||
− | The two new plasmids are respectively named p15. | + | The two new plasmids are respectively named p15.06 and p15.07. |
<br> | <br> | ||
Golden Gate was performed according to the following protocol: | Golden Gate was performed according to the following protocol: | ||
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<th>p15.01</th> | <th>p15.01</th> | ||
<th>p15.02</th> | <th>p15.02</th> | ||
− | <th>p15. | + | <th>p15.06</th> |
− | <th>p15. | + | <th>p15.07</th> |
<th>negative control</th> | <th>negative control</th> | ||
</tr> | </tr> | ||
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Striked a sample of p15.02 lawn on LB + ery (150µg/mL).<br> | Striked a sample of p15.02 lawn on LB + ery (150µg/mL).<br> | ||
− | Striked a sample of p15. | + | Striked a sample of p15.06 lawn on LB + ery (150µg/mL).<br> |
− | Striked a single colony of p15. | + | Striked a single colony of p15.07 on LB + ery (150µg/mL).<br> |
Incubated overnight at 37°C.<br> | Incubated overnight at 37°C.<br> | ||
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<tr> | <tr> | ||
<th>p15.02</th> | <th>p15.02</th> | ||
− | <th>p15. | + | <th>p15.06</th> |
− | <th>p15. | + | <th>p15.07</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
− | Inoculated 5mL LB + ery (150µg/mL) with 3 p15.02 colonies, 4 p15. | + | Inoculated 5mL LB + ery (150µg/mL) with 3 p15.02 colonies, 4 p15.06 colonies and 3 p15.07 colonies, and a negative growth control (non-inoculated LB), incubated overnight at 37°C.<br> |
Miniprep of the g15.49 overnight => p15.01 at 330ng/µL. | Miniprep of the g15.49 overnight => p15.01 at 330ng/µL. | ||
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Restriction Digestion of p15.01 with Eco31I and BbsI (using the digestion protoccol) and PCR purification to remove the enzymes.<br> | Restriction Digestion of p15.01 with Eco31I and BbsI (using the digestion protoccol) and PCR purification to remove the enzymes.<br> | ||
− | Picked 10 single colonies from both p15. | + | Picked 10 single colonies from both p15.06 and p15.07 and launched overnight cultures in LB + ery (150µg/mL).<br> |
Line 1,033: | Line 1,033: | ||
<br><br><b>04/08</b><br><br> | <br><br><b>04/08</b><br><br> | ||
− | Miniprep of all the overnight cultures from clones of p15. | + | Miniprep of all the overnight cultures from clones of p15.06 and p15.07.<br> |
− | Restriction digestion with Eco31I and BbsI of 6 p15. | + | Restriction digestion with Eco31I and BbsI of 6 p15.06 and p15.07 minipreped.<br> |
Restriction analysis on 1% agarose gel: | Restriction analysis on 1% agarose gel: | ||
− | + | <br> | |
<img size="25%" src="https://static.igem.org/mediawiki/2015/b/b7/PB_notebookB2_pic2.jpg"/><br> | <img size="25%" src="https://static.igem.org/mediawiki/2015/b/b7/PB_notebookB2_pic2.jpg"/><br> | ||
<br> | <br> |
Revision as of 10:56, 14 August 2015