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Team:Brasil-SP/Notebook/Protocols
From 2014.igem.org
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| + | <h1 align=center>Experimental Methodology</h1> | ||
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| + | <p>For the assemblies constructuion we addopted the following method:</p> | ||
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| + | <div align=center><img src="https://static.igem.org/mediawiki/2014/f/fc/Metodologia_Brasil-SP.png" width="900" height="400"/></div> | ||
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| + | <br> | ||
| + | <p>We used the pSB1C3 vector from the Biobrick <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>. Using this biobrick we added a red/white selection, where the red colonies are those which do not have the assembly, and the white ones are the correct ones.</p> | ||
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| + | <div align=center><img src="https://static.igem.org/mediawiki/2014/2/25/Red_and_WHite_plate_.jpg" width="700" height="265"/></div> | ||
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<li><a href="https://static.igem.org/mediawiki/2014/3/34/DSM_Medium_Sporulation.pdf">DSM medium for sporulation</a></li> | <li><a href="https://static.igem.org/mediawiki/2014/3/34/DSM_Medium_Sporulation.pdf">DSM medium for sporulation</a></li> | ||
<li><a href="https://static.igem.org/mediawiki/2014/0/00/Medium_LB.pdf">Luria-Bertani (LB) medium</a></li> | <li><a href="https://static.igem.org/mediawiki/2014/0/00/Medium_LB.pdf">Luria-Bertani (LB) medium</a></li> | ||
| - | <li><a href="https://static.igem.org/mediawiki/2014/0/ | + | <li><a href="https://static.igem.org/mediawiki/2014/0/0e/Neubauer_chamber_2.pdf">Cell counting with Neubauer Chamber</a></li> |
| + | <li><a href="https://static.igem.org/mediawiki/2014/a/a2/Germination_Sporulation.pdf">Germination of <i>Bacillus subtilis</i> spores</a></li> | ||
| + | <li><a href="https://static.igem.org/mediawiki/2014/6/63/QuickChange_PCR.pdf">QuickChange PCR</a></li> | ||
| + | <li><a href="https://static.igem.org/mediawiki/2014/3/35/PCR_for_synthesis.pdf">PCR for Synthesis</a></li> | ||
</ul> | </ul> | ||
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<div id="characterizationprotocols" class="row"> | <div id="characterizationprotocols" class="row"> | ||
<h2 align="left"><b>Characterization Protocols</b></h2> | <h2 align="left"><b>Characterization Protocols</b></h2> | ||
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| + | <li><a href="https://static.igem.org/mediawiki/2014/0/00/Cytometry_protocol_characterization.pdf">Cytometry Protocol</a></li> | ||
| + | <li><a href="https://static.igem.org/mediawiki/2014/0/02/Fluorometry_Protocol_Characterization.pdf">Fluorometry Protocol</a></li> | ||
| + | <br><br><br> | ||
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| + | </ul> | ||
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Latest revision as of 23:38, 17 October 2014
Experimental Methodology
For the assemblies constructuion we addopted the following method:
We used the pSB1C3 vector from the Biobrick BBa_J04450. Using this biobrick we added a red/white selection, where the red colonies are those which do not have the assembly, and the white ones are the correct ones.
Lab Protocols
- PCR
- Agarose Gel Electrophoresis and DNA Gel Purification
- PCR Product cloning
- Competent E. coli Cells using Calcium Chloride
- Transformation in E. coli DH5-alpha
- Plasmid preparation by alkaline lysis and SDS
- Cohesive-end assembly cloning
- Restriction Analysis and Digestion in large scale
- Competent Bacillus subtilis cells
- Transformation of Bacillus subtilis
- DSM medium for sporulation
- Luria-Bertani (LB) medium
- Cell counting with Neubauer Chamber
- Germination of Bacillus subtilis spores
- QuickChange PCR
- PCR for Synthesis
