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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Oct
From 2014.igem.org
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| + | <ul> | ||
| + | <li><b><i>pSB1C3_T7_adhA</i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we tried to reclone pSB1A2_T7_<i>adhA</i> into the pSB1C3 backbone.</li> | ||
| + | <li>Because the recloning didn't worked well till now, we decided to restrict the used backbone pSB1C3_RFP with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i> Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i> Spe</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i> Pst</i>I</a> to minimize the chance of religation. | ||
| + | <li>pSB1C3_T7_<i>adhA</i> we cut with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a> | ||
| + | <li>Clean up and Ligation as explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a></li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pB1C3_adhA" target="_blank">rev_pB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_adhA_pSB1C3" target="_blank">fw_adhA_pSB1C3</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65 °C</li> | ||
| + | <li>Bands as expected (~1200 bp)</li> | ||
| + | </ul> | ||
| + | |||
| + | <li>Liquid cultures for a restriction digest were prepared.</li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_T7_<i>adhA</i></li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li> | ||
| + | <ul> | ||
| + | <li>Bands as expected (backbone: ~2,2 kb and insert: ~1,2 kb)</li> | ||
| + | </ul> | ||
| + | </ul></ul> | ||
| + | </ul> </ul> | ||
| + | <br> | ||
| + | |||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 12:50, 16 October 2014
 |
October |
- pSB1C3_T7_adhA
- This week we tried to reclone pSB1A2_T7_adhA into the pSB1C3 backbone.
- Because the recloning didn't worked well till now, we decided to restrict the used backbone pSB1C3_RFP with EcoRI, XbaI, SpeI and PstI to minimize the chance of religation.
- pSB1C3_T7_adhA we cut with EcoRI and PstI
- Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (rev_pB1C3_adhA, fw_adhA_pSB1C3)
- Annealing temperature: 65 °C
- Bands as expected (~1200 bp)
- Liquid cultures for a restriction digest were prepared.
- Plasmid isolation of pSB1C3_T7_adhA
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~1,2 kb)
