Team:Imperial College London/Lab Diaries/Vectors team
From 2010.igem.org
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+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 7 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li>Starting assembly of AmyE vector: | ||
+ | <li>Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe) | ||
+ | <li>Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba) | ||
+ | <li>Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe) | ||
+ | <li>PCR amplification of vector backbone PSB1C3 for the PyrD vector</li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li>Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday) | ||
+ | <li>Gel purification of k70 | ||
+ | <li>Gel purification of k08 | ||
+ | <li>Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up | ||
+ | <li>Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up | ||
+ | <li>Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector | ||
+ | <li>Restriction digestion of pSB1C3 for PyrD (using Eco and Pst) </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li>Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR | ||
+ | <li>Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon | ||
+ | <li>Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li>Transformation of overnight ligations of: k64 and k70, k70 only, Spec and 5' PyrD diff</li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li>Replica plating and colony PCR of: Spec and 5' PyrD diff | ||
+ | <li>Plate wash of: k64 and k70. k70 only was discarded since this was purely for a background check </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
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{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | ||
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* Gel analysis of colony PCR products of Spec and 5' PyrD diff | * Gel analysis of colony PCR products of Spec and 5' PyrD diff | ||
* Annealing of diff P oligos (used for both PyrD and AmyE vectors) | * Annealing of diff P oligos (used for both PyrD and AmyE vectors) | ||
+ | |} | ||
|} | |} | ||
+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 8 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
- | |||
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* Diagnostic digests of minipreps - Two digests : One with Spe & Pst and other with Xba & Spe | * Diagnostic digests of minipreps - Two digests : One with Spe & Pst and other with Xba & Spe | ||
|} | |} | ||
+ | |} | ||
+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 9 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
- | |||
- | |||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | ||
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* Set up ligation reaction K02+dif using 3A method for Transformation on Monday. | * Set up ligation reaction K02+dif using 3A method for Transformation on Monday. | ||
|} | |} | ||
+ | |} | ||
+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 10 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
- | |||
- | |||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | ||
Line 1,199: | Line 1,546: | ||
|} | |} | ||
+ | |} | ||
+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 11 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
- | |||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | ||
Line 1,246: | Line 1,677: | ||
|} | |} | ||
+ | |} | ||
+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 12 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
- | |||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1" | ||
Line 1,293: | Line 1,808: | ||
|} | |} | ||
+ | |} | ||
+ | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
+ | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 13 | ||
+ | |- | ||
+ | | | ||
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <b>Day</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;height:100px;width:150px;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:top;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | <ul> | ||
+ | <li></li> | ||
+ | <li></li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
- | |||
<html> | <html> | ||
<table width="850px" border="0"> | <table width="850px" border="0"> | ||
Line 1,428: | Line 2,027: | ||
* Gel analysis of colony PCR products | * Gel analysis of colony PCR products | ||
* Set up ligation reaction K02+dif using 3A method for Transformation on Monday. | * Set up ligation reaction K02+dif using 3A method for Transformation on Monday. | ||
+ | |} | ||
|} | |} |
Revision as of 00:24, 25 October 2010
Lab Diaries | Overview | Surface Protein Team | XylE Team | Vectors Team | Modelling Team |
Here are the technical diaries for our project. We've split them up into three lab teams and the modelling team. We think it's really important that absolutely anyone can find out what we've been doing. For a really detailed look at what we did, and when, you've come to the right place! |
Objectives |
We are assembling the AmyE and PyrD vectors in order to transform B. Subtilis with our parts. Once completed, these vectors will be reusable and can then be used to introduce any relevant piece of DNA directly into B.subtilis genome. The vectors will be used both for the final assembly and for testing constructs. |
PyrD Vector |
Week 6
Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
Morning |
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Afternoon |
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Thursday, August 12
- Annealing the forward and reverse strands of the dif XP oligo
The forward and reverse strands of the 5' dif site with XbaI and PstI restriction sites on either side have been synthesized separately. The synthesized fragments arrive in solid powder form. These were immediately diluted in ddH2O to obtain a stock concentration of 1 ng/ul. They were then allowed to anneal together by first heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.
Friday, August 13
- Restriction digest of dif XP
After annealing the two strands, the oligo was cut with XbaI and PstI to obtain overhangs that would later ligate with the compatible overhangs of a cut vector.
- PCR purification of the cut dif XP
The cut oligo was PCR purified in order to get rid of any contaminants. PCR purification gets rid of short pieces of DNA which are less than about 40 base pairs.
- Gel Analysis of dif XP
Week 7
Week 7 | Monday | Tuesday | Wednesday | Thursday | Friday |
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Afternoon |
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Monday, August 16
- Restriction digest of 5' ins [K143008]
K143008 is the 5' integration site for the PyrD vector. This will be used as a front insert together with the dif XP for the pSB1C3 vector.
- PCR amplification of pSB1C3
The pSB1C3 vector backbone from the registry was amplified with the use of SB3 and SB2a primers. Submission of parts to the registry requires them to be in a pSB1C3 vector therefore any parts to be submitted will be inserted into this vector.
- PCR purification of pSB1C3
The PCR amplified vector was purified in order to get rid of any contaminants. For example, short pieces of DNA like the primers.
Tuesday, August 17
- Gel extraction and purification of 5' ins ES
The digested 5' ins was first analyzed on the gel to verify it's size and then extracted for purification. The 5' ins was gel purified in order to extract only the relevant piece of DNA.
- Restriction digest of pSB1C3
The pSB1C3 vector was digested so that it would contain compatible overhangs for ligation with inserts.
- PCR purification of pSB1C3 EP
The digested pSB1C3 was re-purified in order to get rid of any contaminant DNA that arose during the digestion.
- Restriction digests of pveg and Spec-T
pveg (promoter and RBS) and Spec-T (Spectinomycin with a terminator) were digested in preparation for 3A assembly.
- Ligation of 5' ins (ES) and dif (XP) with pSB1C3 (EP)
The digested 5' ins and dif (the front inserts) were ligated overnight with pSB1C3 (the vector). A bench ligation and an overnight ligation were set up.
- Transformation of E.Coli with bench ligate
E.Coli was transformed via the chemical method using the bench ligate.
- Gel extraction and purification of pveg and Spec-T
Since these are both inserts they were gel extracted and purified. PCR purification is not carried out for inserts since they are small and would therefore be lost during the process.
Wednesday, August 18
- Replica plating and colony PCR of 5' ins and dif in pSB1C3 (bench ligation)
- Ligation of pveg (ES) and SpecR-T (XP) with pSB1C3 (EP)
pveg and SpecR-T (the front inserts) were ligated overnight with pSB1C3 (the vector).
Thursday, August 19
- Transformation of E.Coli with the overnights ligates
- 5' ins and dif in pSB1C3
- pveg and SpecR-T in pSB1C3
Friday, August 20
- Replica plating and colony PCR of both transformations from yesterday.
- Annealing the forward and reverse strands of the dif P ES oligo
The forward and reverse strands of the 3' dif Pme1 sites with XbaI and PstI restriction sites on either side have been synthesized separately. The synthesized fragments arrive in solid powder form. These were immediately diluted in ddH2O to obtain a stock concentration of 1 ng/ul. They were then allowed to anneal together by heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.
Week 8
Week 8 | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
Morning |
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Afternoon |
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Week 9
Week 9 | Monday | Tuesday | Wednesday | Thursday | Friday |
Morning |
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Afternoon |
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Week 10
Week 10 | Monday | Tuesday | Wednesday | Thursday | Friday |
Morning |
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Afternoon |
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Week 11
Week 11 | Monday | Tuesday | Wednesday | Thursday | Friday |
Morning |
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Afternoon |
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Week 12
Week 12 | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
Morning |
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Afternoon |
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Week 13
Week 13 | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
Morning |
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Afternoon |
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AmyE Vector |
Starting from the top, we are assembling the first two fragments (K14070 and K14064) and K147002 with our oligos to add in a Dif site. Two dif sites on either side of resistance cassettes can be used to later excise antibiotic resistance from our final constructs.
Next Steps:
K14002 and oligos
Next Steps:
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Schedule & Lab Notes |
Week 7 | ||||||||||||||||||||||||||||||||||||
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Week 8 | |||||||||||||||||||||||||||||||||||||||
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Week 9 | |||||||||||||||||||||||||||||||||||||
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Week 10 | |||||||||||||||||||||||||||||||||||||
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Week 11 | |||||||||||||||||||||||||||||||||||||
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Week 12 | |||||||||||||||||||||||||||||||||||||
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Week 13 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
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