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Team:Warsaw/Calendar-Main/22 July 2009
From 2009.igem.org
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<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
| - | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is total volume of ligation mixture prepared 20.07.09</li> | + | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of total volume of ligation mixture prepared 20.07.09</li> |
<li>petri dish were hold in 37°C for 16 hours</li> | <li>petri dish were hold in 37°C for 16 hours</li> | ||
</ul> | </ul> | ||
| Line 50: | Line 50: | ||
<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
| - | <li>Ligation mixture composition: 10 μl digested p53 | + | <li>Ligation mixture composition: |
| - | (Fermentas) | + | <pre>10 μl digested p53 |
| + | 25 μl digested pKS | ||
| + | 5 μl ligation buffer | ||
| + | (Fermentas) | ||
| + | 8 μl MQ water | ||
| + | 2 μl ligase T4</pre> | ||
| + | </li> | ||
<li>Duration of ligation was about 7 hours; reaction was conducted in 16 °c (approximately).</li> | <li>Duration of ligation was about 7 hours; reaction was conducted in 16 °c (approximately).</li> | ||
</ul> | </ul> | ||
| Line 63: | Line 69: | ||
<ul> | <ul> | ||
<li>Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes</li> | <li>Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes</li> | ||
| - | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of half volume of ligation mixture prepared today/li> | + | <li>Detailed protocol of transformation is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of half volume of ligation mixture prepared today</li> |
</ul> | </ul> | ||
| - | |||
| - | |||
| - | <h3><div style="text-align: center;"> | + | <h3><div style="text-align: center;">Isolation of BioBricks from 2008 and 2009 Kit Plates</div></h3> |
<h4>Sebastian</h4> | <h4>Sebastian</h4> | ||
<br/> | <br/> | ||
<p>Task 1:</p> | <p>Task 1:</p> | ||
<ul> | <ul> | ||
| - | <li>Digest of parts BBa_B0032 | + | <li>Digest of following parts</li> |
| + | <ul> | ||
| + | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></li> | ||
| + | <li><a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></li> | ||
| + | <li><a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></li> | ||
| + | <li><a href="http://partsregistry.org/Part:BBa_E0032"><span style="color: black">BBa_E0032</a></li></ul> | ||
</ul> | </ul> | ||
<br/> | <br/> | ||
| Line 79: | Line 88: | ||
<ul> | <ul> | ||
<li>Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI</li> | <li>Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI</li> | ||
| - | <li>Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032): 10ul of plasmid | + | <li>Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032): |
| - | <li>Reaction mixture (BBa_B0032): 5ul of plasmid | + | <pre>10ul of plasmid |
| + | 1ul of each enzyme | ||
| + | 5ul of Fermentas Tango Yellow buffer | ||
| + | 33ul H2O</pre></li> | ||
| + | <li>Reaction mixture (BBa_B0032): | ||
| + | <pre>5ul of plasmid | ||
| + | 0,5ul of each enzyme | ||
| + | 2,5ul of Fermentas Tango Yellow buffer | ||
| + | 16ul H2O</pre></li> | ||
<li>Time of digest - 6h</li> | <li>Time of digest - 6h</li> | ||
</ul> | </ul> | ||
| Line 114: | Line 131: | ||
</ul> | </ul> | ||
<br /> | <br /> | ||
| - | </ | + | </li> |
| + | </ul> | ||
| + | <h3>Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a> operon1_part2 </h3> | ||
| + | |||
| + | <h4>Ania</h4> | ||
| + | <br/> | ||
| + | <p>Tasks:</p> | ||
| + | |||
| + | <ul> | ||
| + | <li>Gel extraction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032"> BBa_B0032</a>- RBS.3 on the pSB1A2 ampicillin resistant plasmid and gel extracion of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012"> - lacI repressor</a> </li> | ||
| + | <li>Overnight ligation of extracted fragments</li> </ul> | ||
| + | </html> | ||
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{{WarNotebookEnd}} | {{WarNotebookEnd}} | ||
Latest revision as of 16:12, 26 September 2009
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Assembly of invasion operon
Marcin
Task 1:
- Prepare chemocompetent bacteria
Detailed protocol
- Incubate 100 ml of liquid bacterial culture until the OD value is 0.55 or little more
- Incubate the bacteria on the ice for 15 minutes
- Centrifuge the bacteria at 4°C for 15 minutes
- Remove the supernatant and add 10 ml of 0.1 M solution of CaCl2
- Gently suspend the bacterial pellet and incubate the bacteria on the ice for 45 minutes
- Centrifuge the bacteria at 4°C for 15 minutes
- Remove the supernatant and add 2 to 5 ml of 0.1 M solution of CaCl2
- Prepate the aliquots of suspended bacteria which contain 100 μl of the solution
- Store the bacteria in -80°C
Assembly of endosomal detection operon
Marcin
Task 1:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Detailed protocol of transformation is described here. The only modification is usage of total volume of ligation mixture prepared 20.07.09
- petri dish were hold in 37°C for 16 hours
Cloning of p53 coding sequence
Marcin
Task 1:
- Cloning p53 coding sequence to pKS plasmid
Methods:
- Ligation mixture composition:
10 μl digested p53 25 μl digested pKS 5 μl ligation buffer (Fermentas) 8 μl MQ water 2 μl ligase T4
- Duration of ligation was about 7 hours; reaction was conducted in 16 °c (approximately).
Task 2:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of transformation is described here. The only modification is usage of half volume of ligation mixture prepared today
Isolation of BioBricks from 2008 and 2009 Kit Plates
Sebastian
Task 1:
Methods:
- Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI
- Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032):
10ul of plasmid 1ul of each enzyme 5ul of Fermentas Tango Yellow buffer 33ul H2O
- Reaction mixture (BBa_B0032):
5ul of plasmid 0,5ul of each enzyme 2,5ul of Fermentas Tango Yellow buffer 16ul H2O
- Time of digest - 6h
Task 2:
- Electrophoresis and gel-out
Methods:
- Electrophoresis of all samples volume https://static.igem.org/mediawiki/2009/a/ab/Trawienia22-07.JPG
- Order of samples: (1,2) BBa_E0032, (3,4) BBa_C0051, (5) generuler DNA Mix, (6,8,9) BBa_C0040, (7) BBa_B0032
- Gel-out of samples with A&A Biotechnology Kit
- Electrophoresis of the samples after Gel-out https://static.igem.org/mediawiki/2009/8/8a/Gel-out.JPG
- Order of samples: BBa_B0032, BBa_C0040, BBa_C0051, BBa_E0032, generuler DNA mix
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Transformation verification
Methods:
- In order to save on resources colonies that potentially contain the mgtc promoter (the white ones) were marked and the entire dish returned to 37°C for the next 24h.
Construction of K177012 operon1_part2
Ania
Tasks:
- Gel extraction of BBa_B0032- RBS.3 on the pSB1A2 ampicillin resistant plasmid and gel extracion of - lacI repressor
- Overnight ligation of extracted fragments
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