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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep
From 2014.igem.org
(Difference between revisions)
| Line 300: | Line 300: | ||
<ul> | <ul> | ||
<li>We tried to assemble both parts of <i>sap</i>.</li> | <li>We tried to assemble both parts of <i>sap</i>.</li> | ||
| - | + | <ul> | |
| - | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> on the pJet plasmid for <i>sap_2</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#sap_2_fwd" target="_blank">sap_2_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#sap_2_rev" target="_blank">sap_2_rev</a>)</li> | |
| - | + | <ul> | |
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~1500 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>sap_1</i>, <i>sap_2</i> and pSB1C3</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~3000 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <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#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li> | ||
| + | <ul> | ||
| + | <li>Bands as expected (~2000 bp (backbone) and ~2700 bp (insert))</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <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#RestrictionEnzymes" target="_blank"><i>EcoR</i>V</a></li> | ||
| + | <ul> | ||
| + | <li>Bands as expected (~1500 bp and ~3200 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | </ul> | ||
| + | </ul> | ||
| + | </div> | ||
| + | </div> | ||
</div> | </div> | ||
| - | |||
</div> | </div> | ||
Revision as of 12:27, 2 October 2014
 |
September |
 |
- csoS1-4
- We tried to amplify csoS1-4 (shell proteins) for a fusion with GFP.
- PCR amplification (fw-GFP-csoS1B, rv-rv-GFP-csoS1A)
- Annealing temperature: 54 °C
- Bands as expected (~4000 bp (~1800 bp csoS1-4, ~2200 bp pSB1C3))
- PCR products were purified
- csoS1-4 and GFP
- We tried to assemble the shell proteins of the carboxysome and GFP.
- Gibson Assembly with pSB1C3_csoS1-4 and GFP
- Transformation with electrocompotetent cells
- Colony PCR (fw-csoS1A-GFP, rv-csoS1B-GF)
- Annealing temperature: 68 °C
- Bands as expected (~1000 bp)
- Plasmid isolation of pSB1C3_csoS1-4_GFP
- Restriction digestion with NotI
- Bands (as expected (~2000 bp and ~2400 bp)
- csoS1-4_GFP and T7
- We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promotor.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, rv_csoS4A_PstI)
- Annealing temperature: 55 °C
- Bands as expected (~320 bp)
- can and csoS1-4
- This week we tried to find positive clones of our transformation.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2100 bp and ~3300 bp)
- can_csoS1-4 and csoS1D
- We tried to assemble our pSB1C3_can_csoS1-4 construct with csoS1D.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~4300 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4_csoS1D
- Restriction digestion with NotI
- Bands as expected (~2000 bp and ~4000 bp)
- Hneap and T7
- This week we tried to assemble the T7 promotor with the Hneap.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Hneap and T7
- We tried to find positiv clones of pSB3A2_T7_Hneap.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2000 bp)
- Plasmid isolation of pSB1A2_T7_Hneap
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp and ~1800 bp)
- sap
- We tried to assemble both parts of sap.
- PCR amplification on the pJet plasmid for sap_2 (sap_2_fwd, sap_2_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1500 bp)
- Gibson Assembly with sap_1, sap_2 and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3000 bp)
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp (backbone) and ~2700 bp (insert))
- Restriction digestion with EcoRV
- Bands as expected (~1500 bp and ~3200 bp)
