Team:TU-Eindhoven/Protocols

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<h3>Standard protocols and modifications</h3>
<h3>Standard protocols and modifications</h3>
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<h3>BioBrick<sup>TM</sup> construction</h3>
<h3>BioBrick<sup>TM</sup> construction</h3>
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<p>Many protcols for assembling BioBricks<sup>TM</sup> are already available, like Standard Assembly and 3A-assembly. These are suited for the assembly of standardized bricks, however, we created our BioBricks<sup>TM</sup> out of non-standard material and when working with yeast we used non-standard vectors to carry the protein coding sequences. In other words, most of our cloning steps had to be done the old-fashioned way and consequently the yields were lower than with standard assembly. The solution was to use more DNA and longer incubation for digestion. This guaranteed a proper yield at the end of the process. The detail can be found in our [[File:BioBrick_construction|high-efficiency BioBrick<sup>TM</sup> assembly protocol]]. This includes digestion, ligation and transformation.</p>
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<p>Many protcols for assembling BioBricks<sup>TM</sup> are already available, like Standard Assembly and 3A-assembly. These are suited for the assembly of standardized bricks, however, we created our BioBricks<sup>TM</sup> out of non-standard material and when working with yeast we used <span class="red">non-standard vectors</span> to carry the protein coding sequences. In other words, most of our <span class="red">cloning </span>steps had to be done the <span class="red">old-fashioned way</span> and consequently the yields were lower than with standard assembly. The solution was to use more DNA and longer incubation for digestion. This guaranteed a proper yield at the end of the process. The detail can be found in our <html><a href="https://static.igem.org/mediawiki/2012/d/dd/BioBrickprotocol.pdf" target="_blank">high-efficiency BioBrick<sup>TM</sup> assembly protocol</a></html>. This includes <span class="red">digestion, ligation and transformation</span>.</p>
<h3>Yeast transformation</h3>
<h3>Yeast transformation</h3>
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<p>The principal protocol for yeast transformation was Gietz and Schiestl (2007)<html><a href="#ref_gietz_2007" name="text_gietz_2007"><sup>[1]</sup></a></html>. It promised high transformation efficiency, however, in combination with our yeast the yield was a factor thousand lower than expected. The solution was to increase the amount of DNA used for transformation up to a whopping 3 ug and to work with freshly cultured yeast in mid-log phase instead of vials of frozen competent cells. For more details, please refer to the [[File:yeast_transformation.pdf|modified yeast transformation protocol]].</p>
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<p>The principal protocol for yeast transformation was Gietz and Schiestl (2007)<html><a href="#ref_gietz_2007" name="text_gietz_2007"><sup>[1]</sup></a></html>. It promised high transformation efficiency, however, in combination with our yeast the <span class="red">yield</span> was a factor thousand <span class="red">lower </span> than expected. The solution was to <span class="red">increase the amount of DNA</span> used for transformation up to a whopping 3 &micro;g and to work with freshly cultured yeast in mid-log phase instead of vials of frozen competent cells. For more details, please refer to the <html><a href="https://static.igem.org/mediawiki/2012/d/da/Yeast_transformation.pdf" target="_blank">modified yeast transformation protocol</a></html>.</p>
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<h3>Pretty Standard Protocols (PSP<sup>TM</sup>)</h3>
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<h3>Pretty Standard Protocols (PSP)</h3>
<p>The following protocols were regularly used in the lab and are referred to implicitly in the weekly notebook pages.
<p>The following protocols were regularly used in the lab and are referred to implicitly in the weekly notebook pages.
<ul>
<ul>
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<li>Media: [[LB]], [[YPD]] [[SDCAA]], [[SC]].</li>
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<li>Media:  
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<li>Culture: [[E. coli]], [[S. cerevisiae]].</li>
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<html>
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<li>Cryopreservation: [[E. coli]], [[S. cerevisiae]].</li>
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<a href="https://static.igem.org/mediawiki/2012/d/d7/LB.pdf" target="_blank">LB</a>,
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<li>Expression of protein: From [[pET|pET-vector]], from [[pYES2/pYES3]].</li>
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<a href="https://static.igem.org/mediawiki/2012/b/b9/YPD.pdf" target="_blank">YPD</a>,
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<li>QIAgen kits: [[Miniprep]], [[gel extration]], [[PCR-purfication]].</li>
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<a href="https://static.igem.org/mediawiki/2012/7/77/SDCCA.pdf" target="_blank">SDCAA</a>,
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<li>Cloning: [[Colony PCR]].</li>
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<a href="https://static.igem.org/mediawiki/2012/b/b7/SC.pdf" target="_blank">SC</a>,
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<a href="https://static.igem.org/mediawiki/2012/2/2a/Phosphate_buffer.pdf" target="_blank">Phosphate buffer</a>.</li>
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<li>Culture:  
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<a href="https://static.igem.org/mediawiki/2012/0/08/Culture_E._coli.pdf" target="_blank">E. coli</a>,
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<a href="https://static.igem.org/mediawiki/2012/0/0b/Culture_S._cerevisiae.pdf" target="_blank">S. cerevisiae</a>.</li>
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<li>Cryopreservation:  
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<a href="https://static.igem.org/mediawiki/2012/5/50/Cryopresevation_E._coli.pdf" target="_blank">E. coli</a>,
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<a href="https://static.igem.org/mediawiki/2012/c/cd/Cryopresevation_S._cerevisiae.pdf" target="_blank">S. cerevisiae</a>.</li>
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<li>Expression of protein:  
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From <a href="https://static.igem.org/mediawiki/2012/d/de/PET_system_manual.pdf" target="_blank">pET-vector</a>,
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from <a href="https://static.igem.org/mediawiki/2012/d/da/PYES2%2C_pYES3_manual.pdf" target="_blank">pYES2/pYES3</a>.</li>
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<li>QIAgen kits:  
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<a href="https://static.igem.org/mediawiki/2012/8/86/QIAprep_miniprep.pdf" target="_blank">Miniprep</a>,
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<a href="https://static.igem.org/mediawiki/2012/1/13/QIAgen_gel_extraction.pdf" target="_blank">gel extraction</a>,
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<a href="https://static.igem.org/mediawiki/2012/a/a3/QIAquick_PCR-purification.pdf" target="_blank">PCR-purification</a>.</li>
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<li>Cloning:  
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<a href="https://static.igem.org/mediawiki/2012/6/66/Colony_PCR.pdf" target="_blank">Colony PCR</a>.</li>
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</ul>
</ul>
</p>
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'''References'''
'''References'''
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<html>
<html>
<ul>
<ul>
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<li><a href="#text_gietz_2007" name="ref_gietz_2007">[1]</a>Gietz and Schiestl, ''High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method'', Nature Protocols (2007) vol. 2, issue 1, pp. 31-34</li> </ul> </html>
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<li><a href="#text_gietz_2007" name="ref_gietz_2007">[1]</a>R. Gietz and R. Schiestl, High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nature Protocols 2: 31-34, (2007)</li> </ul> </html>
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Latest revision as of 02:07, 27 September 2012