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Team:Warsaw/Calendar-Main/25 July 2009
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(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> <h4>Marcin</h4> <br/> <p>Task 1:</p> <ul> <li...) |
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<ul><li>Reaction mixture composition: 0.5 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl PstI (Fermentas), 2 μl Buffer Tango (Fermentas), 16.5 μl MQ water</li></ul> | <ul><li>Reaction mixture composition: 0.5 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl PstI (Fermentas), 2 μl Buffer Tango (Fermentas), 16.5 μl MQ water</li></ul> | ||
<li>The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.</li> | <li>The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.</li> | ||
| - | <li>In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids</li> | + | <li>In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids</li></ul> |
<br/> | <br/> | ||
<p><b>Comment:</b></p> | <p><b>Comment:</b></p> | ||
| - | <p>All isolated plasmids have not insert. The cloning must be performed another time</p> | + | <p>All isolated plasmids have not insert. The cloning must be performed another time</p><br/> |
| - | + | <p>Task 2:</p> | |
| + | <ul> | ||
| + | <li>Restriction digest of biobricks</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p><strong>Comment:</strong></p> | ||
| + | <p>Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:</p> | ||
| + | <p> <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span></p> | ||
| + | <p> <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_c0040</a></span></p> | ||
| + | <p> <a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span></p> | ||
| + | <p> <a href="http://partsregistry.org/Part:BBa_E0032"><span style="color: black">BBa_E0032</a></span></p> | ||
| + | <br/> | ||
| + | <p>Methods:</p><ul> | ||
| + | <li>Digest of BBa_B0032 using SpeI and PstI</li> | ||
| + | <ul><li>Reaction mixture composition: 10 μl purified plasmid DNA product, 0.5 μl SpeI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water</li></ul> | ||
| + | <li>Digest of other biobricks using PstI and XbaI</li> | ||
| + | <ul><li>Reaction mixture composition: 10 μl purified plasmid DNA product, 1 μl XbaI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water</li></ul> | ||
| + | <li>Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80°C for 20 minutes</li> | ||
| + | </ul></li><br/> | ||
| + | <p>Task 3:</p> | ||
| + | <ul> | ||
| + | <li>Ligations of biobricks</li> | ||
| + | </ul> | ||
| + | <p>Methods:</p><ul> | ||
| + | <li>Ligation mixture composition: 8 μl digested plasmid with RBS, 9 μl digested CDS, 2 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4 (Fermentas)</li> | ||
| + | <li>Negative control mixture composition: 8 μl digested plasmid with RBS, 2 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4 (Fermentas), 9 μl MQ water</li> | ||
| + | <li>Duration of ligation was about 12 hours</li> | ||
| + | </ul> | ||
| + | <br/> | ||
</html> | </html> | ||
Revision as of 18:13, 2 August 2009
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Assembly of endosomal detection operon
Marcin
Task 1:
- Digest of isolate plasmids with ligated biobricks to verify the success of ligation
Methods:
- Digest of isolate plasmids using XbaI and PstI
- Reaction mixture composition: 0.5 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl PstI (Fermentas), 2 μl Buffer Tango (Fermentas), 16.5 μl MQ water
- The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids
Comment:
All isolated plasmids have not insert. The cloning must be performed another time
Task 2:
- Restriction digest of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:
Methods:
- Digest of BBa_B0032 using SpeI and PstI
- Reaction mixture composition: 10 μl purified plasmid DNA product, 0.5 μl SpeI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water
- Digest of other biobricks using PstI and XbaI
- Reaction mixture composition: 10 μl purified plasmid DNA product, 1 μl XbaI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water
- Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80°C for 20 minutes
Task 3:
- Ligations of biobricks
Methods:
- Ligation mixture composition: 8 μl digested plasmid with RBS, 9 μl digested CDS, 2 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4 (Fermentas)
- Negative control mixture composition: 8 μl digested plasmid with RBS, 2 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4 (Fermentas), 9 μl MQ water
- Duration of ligation was about 12 hours
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