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Team:Warsaw/Calendar-Main/20 August 2009
From 2009.igem.org
(Difference between revisions)
(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3>Amplyfing of Pho sequence</h3> <h4>Justyna</h4> <p>Methods</p> <br/> <ul> <li>PCR reaction mix:</li> <br/> per 50μl: <pre> 5.0 ...) |
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</ul> | </ul> | ||
<br> | <br> | ||
| - | <h3>Cloning Pho into | + | <h3>Cloning Pho into pSB1A3 plasmid</h3> |
<h4>Justyna</h4> | <h4>Justyna</h4> | ||
<p>Task 1:</p> | <p>Task 1:</p> | ||
| Line 62: | Line 62: | ||
<p>Methods:</p> | <p>Methods:</p> | ||
<ul><li>Pho PCR products were isolated from agarose gel using A&A Gel-Out kit.</li> | <ul><li>Pho PCR products were isolated from agarose gel using A&A Gel-Out kit.</li> | ||
| + | <br> | ||
<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2009/3/39/Phoafergeloutopis.jpg"></center> | <img src="https://static.igem.org/mediawiki/2009/3/39/Phoafergeloutopis.jpg"></center> | ||
| + | <br> | ||
<li>The arrow points the right and best isolated product</li> | <li>The arrow points the right and best isolated product</li> | ||
</ul> | </ul> | ||
| - | <ul> | + | <ul><li>pSB1A3 plasmid was previously prepared as described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/14_August_2009">here</a> </li> |
</ul> | </ul> | ||
| Line 73: | Line 75: | ||
<br><br><br> | <br><br><br> | ||
</ul> | </ul> | ||
| + | </html> | ||
| + | |||
| + | <html> | ||
| + | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | ||
| + | <h4>Marcin</h4> | ||
| + | <br/> | ||
| + | <p>Task 1:</p> | ||
| + | <ul><li>Gel-out of <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li></ul> | ||
| + | <p>Methods:</p><ul> | ||
| + | <li>sample was inactivated via heating in 80 °C for 20 minutes | ||
| + | <li>Gel-out was performed using the EurX gel-out kit according to the manual</a></li></ul> | ||
| + | <p>Results:</p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2009/5/5c/C0040%2BRBS_20_08_09.png" width="25%" heigth="25%"</center> | ||
| + | <font face="Times New Roman" size="3"><p><div style="text-align: center;">Result of the digestion</div></p></font> | ||
| + | <br/> | ||
| + | <p>Task 2:</p> | ||
| + | <ul> | ||
| + | <li>Ligate <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> to <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid with <a href="http://partsregistry.org/Part:BBa_R0080"><span style="color: black">BBa_R0080</a></span></li></ul> | ||
| + | <p>Methods:</p><ul> | ||
| + | <li>Reaction mixture composition:</li><pre> | ||
| + | 10 μl insert | ||
| + | 7 μl vector | ||
| + | 2.3 μl Buffer Tango (Fermentas) | ||
| + | 3 μl dNTPs mixture (EurX) | ||
| + | 1 μl T4 ligase (Fermentas) | ||
| + | </pre> | ||
| + | <li>Ligation was performed for about 12 hours and it was subsequently thermally inactivated</li></ul> | ||
| + | <br/> | ||
| + | <p>Task 3:</p><ul><li>Transformation of chemocompetent E. coli strain DH5&alpha</ul></li> | ||
| + | <p>Constructs to transform:</p></ul> | ||
| + | <ul> | ||
| + | <li>p53 CDS + <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid </li> | ||
| + | <li>cro CDS + <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid</li></ul> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>.</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | |||
| + | <html> | ||
| + | <h3><div style="text-align: center;">Making of the plac-RBS-llo part</div></h3> | ||
| + | <h4>Jarek</h4> | ||
| + | <br /> | ||
| + | <p>Tasks:</p> | ||
| + | <ul> | ||
| + | <li>Separation of restriction fragments in 0,8% agarose gel </li> | ||
| + | <li>Digestion of 5 samples of plasmid DNA acquired on 19 August with PvuII and PstI restriction endonucleases</li> | ||
| + | </ul> | ||
| + | <br /> | ||
| + | <h3><div style="text-align: center;">Cloning switch 1 regulatory parts [ | ||
| + | <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>-PcI.RBS.LacI, | ||
| + | <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>-PcI.RBS.LacI.PcI.RBS.RFP.terminator, | ||
| + | <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>-PLacI.RBS.cI.terminator, | ||
| + | <a href="http://partsregistry.org/Part:BBa_K177038">K177038</a>-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator | ||
| + | ] | ||
| + | into two compatible low copy number plasmids of different antibiotic resistance</div></h3> | ||
| + | <h4>Ania</h4> | ||
| + | <br/> | ||
| + | <p>Tasks:</p> | ||
| + | <ul> | ||
| + | <li> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <br/> | ||
</html> | </html> | ||
Latest revision as of 22:55, 13 September 2009
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Amplyfing of Pho sequence
Justyna
Methods
- PCR reaction mix:
per 50μl:
5.0 μl - 10 x buffer (20mM MgSO4) 3.5 μl - dNTP mix (5mM) 2.5 μl - primer PhoF 2.5 μl - primer PhoR2 3.75μl - DMSO 2.0 μl - template DNA 28.25μl - mQ water 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)
Thermal cycling conditions for PCR:
94°C, 1 min 0s 94°C, 0 min 15s 55°C, 0 min 30s 68°C / 72°C (either), 1 min 0s cycles 1-25 68°C / 72°C (either), 7 min 0s 4°C, indefinite
Results:
Cloning Pho into pSB1A3 plasmid
Justyna
Task 1:
- Gel-out Pho PCR product
Methods:
- Pho PCR products were isolated from agarose gel using A&A Gel-Out kit.
- The arrow points the right and best isolated product
- pSB1A3 plasmid was previously prepared as described here
Assembly of endosomal detection operon
Marcin
Task 1:
Methods:
- sample was inactivated via heating in 80 °C for 20 minutes
- Gel-out was performed using the EurX gel-out kit according to the manual
Results:
Result of the digestion
Task 2:
Methods:
- Reaction mixture composition:
10 μl insert 7 μl vector 2.3 μl Buffer Tango (Fermentas) 3 μl dNTPs mixture (EurX) 1 μl T4 ligase (Fermentas)
Task 3:
- Transformation of chemocompetent E. coli strain DH5&alpha
Constructs to transform:
Methods:
- Detailed protocol of transformation is described here.
Making of the plac-RBS-llo part
Jarek
Tasks:
- Separation of restriction fragments in 0,8% agarose gel
- Digestion of 5 samples of plasmid DNA acquired on 19 August with PvuII and PstI restriction endonucleases
Ania
Tasks:
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