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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Sep
From 2014.igem.org
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| + | <ul> | ||
| + | <li><b><i>pSB1A2_T7_<i>adhA</i></i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we tried to contruct the pSB1A2_T7_<i>adhA</i>-Plasmid </li> | ||
| + | |||
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | ||
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<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> | <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> | <ul> | ||
| - | <li> | + | <li><i>adhA</i></li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
</ul> | </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#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65 °C</li> | ||
| + | <li>Bands not as expected (~ 1200 bp)</li> | ||
| + | </ul></ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we tried to contruct the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i>-Plasmid by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li> | ||
| + | <ul> | ||
| + | <li>Upstream part pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> (digested 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>Spe</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li> | ||
| + | </ul> | ||
| + | <li>Downstream part 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>adhA</i></li> | ||
| + | </ul> | ||
| + | <li>Destination part pSB1C3_RFP (digested 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>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </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#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65°C</li> | ||
| + | <li>Bands not as expected (~3000 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul><li>Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> </li></ul>. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. | ||
| + | </ul> | ||
| + | </ul> | ||
</div> | </div> | ||
Revision as of 16:50, 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)
- pSB1A2_T7_adhA
- This week we tried to contruct the pSB1A2_T7_adhA-Plasmid
- BioBrick Assembly (Suffix)
- Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
- Annealing temperature: 65 °C
- Bands not as expected (~ 1200 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)
- Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the PCR purification
