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Team:Warsaw/Calendar-Main/23 July 2009
From 2009.igem.org
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| - | <h3><div style="text-align: center;"> | + | <h3><div style="text-align: center;">Cloning of mitochondrial targeting signal</div></h3> |
<h4>Sebastian</h4> | <h4>Sebastian</h4> | ||
<br/> | <br/> | ||
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<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
| - | <li>Reaction mixture: 1ul pAcGFP1-Mito (5ng) | + | <li>Reaction mixture: |
| - | <li>PCR program: 5min 95C | + | <pre>1ul pAcGFP1-Mito (5ng) |
| + | 1ul dNTPs (2mM) | ||
| + | 1ul L-primer (5pmol/ul) | ||
| + | 1ul R-primer (5pmol/ul) | ||
| + | 1ul Pfu buffer | ||
| + | 4,5ul ddH<sub>2</sub>0 | ||
| + | 0,5ul Pfu DNA polymerase</pre></li> | ||
| + | <li>PCR program: | ||
| + | <pre>5min 95C | ||
| + | 1min 95C | ||
| + | 1min gradient 48-60C | ||
| + | 1min 72C | ||
| + | go to 2-nd step (30x) | ||
| + | 5min 72C</pre></li> | ||
</ul> | </ul> | ||
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<li>Prepare the bacterial cultures for plasmid isolation</li> | <li>Prepare the bacterial cultures for plasmid isolation</li> | ||
</ul> | </ul> | ||
| - | <p>Preparation of bacterial cultures</p> | + | <p>Preparation of bacterial cultures</p><ul> |
<li>Prepare LB medium with kanamycin</li> | <li>Prepare LB medium with kanamycin</li> | ||
<li>Add 3.5 ml of the medium to the probes</li> | <li>Add 3.5 ml of the medium to the probes</li> | ||
<li>Add one bacterial colony to each probe</li> | <li>Add one bacterial colony to each probe</li> | ||
| - | <li>Breed the bacteria about 7 hours</li> | + | <li>Breed the bacteria about 7 hours</li></ul> |
<h3><div style="text-align: center;">Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a> operon1_part2 </div></h3> | <h3><div style="text-align: center;">Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a> operon1_part2 </div></h3> | ||
Latest revision as of 16:15, 26 September 2009
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Cloning of mitochondrial targeting signal
Sebastian
Task 1:
- PCR with gradient temperature of anniling
Methods:
- Reaction mixture:
1ul pAcGFP1-Mito (5ng) 1ul dNTPs (2mM) 1ul L-primer (5pmol/ul) 1ul R-primer (5pmol/ul) 1ul Pfu buffer 4,5ul ddH20 0,5ul Pfu DNA polymerase
- PCR program:
5min 95C 1min 95C 1min gradient 48-60C 1min 72C go to 2-nd step (30x) 5min 72C
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Transformant selection
Methods:
- The colonies that remained white after 48h were picked up and transferred to a new petri dish.
- Liquid cultures were established along the way for plasmid purification.
- Both the dish and the liquid cultures were incubated at 37°C overnight.
Assembly of endosomal detection operon
Marcin
Task 1:
- Prepare the bacterial cultures for plasmid isolation
Preparation of bacterial cultures
- Prepare LB medium with kanamycin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- Breed the bacteria about 7 hours
Construction of K177012 operon1_part2
Ania
Tasks:
- Transformation of the chemocompetent cells with the ligation of BBa_B0032- RBS.3 on the pSB1A2 ampicillin resistant plasmid and - lacI repressor
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