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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Sep
From 2014.igem.org
(Difference between revisions)
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<li>Bands not as expected (~ 1200 bp)</li> | <li>Bands not as expected (~ 1200 bp)</li> | ||
</ul></ul> | </ul></ul> | ||
| + | </ul> | ||
| + | BioBrick Assembly: | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | ||
| + | <ul> | ||
| + | <li>Backbone pSB1A2_T7(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li>pSB1A2_T7</li> | ||
| + | </ul> | ||
| + | <li>Insert pSB1K3_<i>adhA</i>(digested with <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>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>pSB1A2_T7_<i>adhA</i></i></li> | ||
| + | </ul> | ||
| + | </ul> | ||
</ul> | </ul> | ||
Revision as of 16:30, 15 October 2014
 |
September |
 |
- adhA
- This week we tried to verify the positive cutures that were identified last week by a restriction digest
- Plasmid isolation of pSB1K3_adhA
- Restriction digestion with EcoRI and PstI of pSB1K3_adhA
- Bands as expected (~1200 bp)
- Successful sequencing
Agarose gel from the restriction digestion with EcoRI and PstI. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- T7_alsS_ilvC_ilvD_kivD
- This week we tried to verify the positive cutures that were identified last week by a restriction digest
- Plasmid isolation of pSB1A2_T7_alsS_ilvC_ilvD_kivD
- Restriction digestion with EcoRI and PstI of pSB1A2_T7_alsS_ilvC_ilvD_kivD
- Bands not as expected (~7000bp)
- pSB1K3_alsS_ilvC_ilcD_kivD_adhA
- This week we tried to combine the pSB1K3_alsS_ilvC_ilvD_kivD with adhA
- BioBrick Assembly (Suffix)
- Colony PCR (fw-ilvD-kivD, rev-pSB1C3-adhA)
- Annealing temperature: 65 °C
- Bands not as expected (~ 3800 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD
- This week we tried to reclone alsS_ilvC_ilvD_kivD into pSB1C3
- Restriction digestion of pSB1K3_alsS_ilvC_ilvD_kivD and pSB1C3_RFP with EcoRI and PstI
- Bands as expected
(~ 7500 bp for alsS_ilvC_ilvD_kivD and ~ 2200 bp for pSB1C3) - Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvD_kivD, rv_pSB1C3_kivD)
- Annealing temperature: 65 °C
- Bands not as expected (~1800 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD_adhA
- This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly
- Colony PCR (fw_ilvD_kivD , rev_pSB1C3_adhA)
- Annealing temperature: 65°C
- Bands not as expected (~3000 bp)
- pSB1C3_alsS_ilvC_ilvD_kivD
- This week we tried to reclone alsS_ilvC_ilvD_kivD into pSB1C3
- Restriction digestion of pSB1K3_alsS_ilvC_ilvD_kivD and pSB1C3_RFP with EcoRI and PstI
- Bands as expected
(~ 7500 bp for alsS_ilvC_ilvD_kivD and ~ 2200 bp for pSB1C3) - Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvD_kivD, rv_pSB1C3_kivD)
- Annealing temperature: 65 °C
- Bands not as expected (~1800 bp)
- pSB1C3_adhA
- This week we tried to reclone adhA into pSB1C3
- Restriction digestion of pSB1K3_adhA and pSB1C3_RFP with EcoRI and PstI
- Bands as expected
(~ 1200 bp for adhA and ~ 2200 bp for pSB1C3) - Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands not as expected (~ 1200 bp)
- BioBrick Assembly (Suffix)
