Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB2"
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Ligation of annealed oligos o15.011 and o15.012 with digested pSIP411new and o15.072 and o15.073 with digested pKV6 using the following protocol | Ligation of annealed oligos o15.011 and o15.012 with digested pSIP411new and o15.072 and o15.073 with digested pKV6 using the following protocol | ||
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<td><b>Ligation Protocol</b><br> | <td><b>Ligation Protocol</b><br> | ||
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Transformation of p15.01, p15.02, pKV6(miniprep), pSIP411(miniprep) and a negative control (no DNA) in NEB DH5α chemically competent cells<br/> | Transformation of p15.01, p15.02, pKV6(miniprep), pSIP411(miniprep) and a negative control (no DNA) in NEB DH5α chemically competent cells<br/> | ||
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<td><b>Heat Shock transformation protocol</b><br/> | <td><b>Heat Shock transformation protocol</b><br/> | ||
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<b>Transformation results</b> | <b>Transformation results</b> | ||
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<th>Transformed product</th> | <th>Transformed product</th> | ||
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Preparation of DH5α Electrocompetent cells, 50 tubes of 100µL. | Preparation of DH5α Electrocompetent cells, 50 tubes of 100µL. | ||
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<td><b>Electrocompetent Cells Preparation Protocol</b> | <td><b>Electrocompetent Cells Preparation Protocol</b> | ||
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Miniprep of the 3 overnight cultures of p15.01 transformants T1, T2 and T3(respective concentration: 204.8, 160.7 and 267.7 ng/µL).<br> | Miniprep of the 3 overnight cultures of p15.01 transformants T1, T2 and T3(respective concentration: 204.8, 160.7 and 267.7 ng/µL).<br> | ||
Digestion of pKV6(negative control) and p15.01 with BbsI and Eco31I to control the insertion of the two BbsI sites in p15.01. | Digestion of pKV6(negative control) and p15.01 with BbsI and Eco31I to control the insertion of the two BbsI sites in p15.01. | ||
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<td><b>Analytical digestion protocol</b><br> | <td><b>Analytical digestion protocol</b><br> | ||
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Transformation of DH5α by electroporation with the previously ligated p15.01 and p15.02.<br> | Transformation of DH5α by electroporation with the previously ligated p15.01 and p15.02.<br> | ||
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<td><b>Electroporation Protocol</b><br> | <td><b>Electroporation Protocol</b><br> | ||
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<b>Transformation results</b> | <b>Transformation results</b> | ||
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<th>Transformed product</th> | <th>Transformed product</th> | ||
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<b>Transformation results</b> | <b>Transformation results</b> | ||
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<th>Transformed product</b></td> | <th>Transformed product</b></td> | ||
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All the PCR purified product was run on an 1% agarose gel, and gel extraction was performed on the band of approximative size 3kb, according to the following protocol.<br> | All the PCR purified product was run on an 1% agarose gel, and gel extraction was performed on the band of approximative size 3kb, according to the following protocol.<br> | ||
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<tr> | <tr> | ||
<td><b>Gel Extraction Protocol with QIAGEN kit</b><br> | <td><b>Gel Extraction Protocol with QIAGEN kit</b><br> |
Revision as of 09:18, 13 August 2015