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Team:Warsaw/Calendar-Main/4 August 2009
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(New page: {{WarNotebook}} <!-- do not edit above me! --> ===<div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div>=== '''Monika''' <!-- do not remove th...) |
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===<div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div>=== | ===<div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div>=== | ||
| - | + | __NOEDITSECTION__ | |
'''Monika''' | '''Monika''' | ||
| + | Tasks | ||
| + | *Plasmid assembly | ||
| + | |||
| + | |||
| + | Methods | ||
| + | *The plasmid digest mix contained: 4μl purified plasmid, 2μl Tango buffer (Fermentas), 0.5μl SpeI enzyme, 0.5μl XbaI enzyme, the solution was topped up with H2O to the final volume of 20μl. | ||
| + | *The mgtc promoter digest mix contained: 12μl purified promoter, 3μl Tango buffer (Fermentas), 0.5μl SpeI enzyme, 0.5μl XbaI enzyme, the solution was topped up with H2O to the final volume of 30μl. | ||
| + | *The digest was carried out in 37°C for 3h, but 1μl CIAP enzyme was added 1h before end to mgtc promoter digest mix 1h before the end of incubation. Then enzymes were inactivated in 80°C for 20min. | ||
| + | |||
| + | |||
| + | <html> | ||
| + | |||
| + | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | ||
| + | <h4>Marcin</h4> | ||
| + | <br/> | ||
| + | <p>Task 1:</p> | ||
| + | <ul> | ||
| + | <li>Isolate the plasmid containing <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> and <a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span> from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis</li> | ||
| + | <p>Methods:</p> | ||
| + | <li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf" here>here</a></li></ul> | ||
| + | <br/> | ||
| + | <p>Task 2:</p> | ||
| + | <ul> | ||
| + | <li>Digest of isolate plasmids with ligated biobricks to verify the success of ligation </li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Methods:</p><ul> | ||
| + | <li>Digest of isolate plasmids using XbaI and PstI</li> | ||
| + | <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>In the next step reaction mixtures were loaded into the agarose gel to analyse restriction pattern of the plasmids</li></ul> | ||
| + | <br/> | ||
| + | <p>Results:</p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2009/8/85/C0040_digest_04_08_09.png" height="35%" width="35%"></center> | ||
| + | <font face="Times New Roman" size="3"><p><div style="text-align: center;">verification of the restriction patterns</div></p></font> | ||
| + | <p><b>Comment:</b></p> | ||
| + | <p>All isolated plasmids do not have insert. The cloning must be performed another time</p><br/> | ||
| + | <p>Task 3:</p> | ||
| + | <br/> | ||
| + | <ul> | ||
| + | <li> Another Cloning of this biobrick</li> | ||
| + | </ul> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li>Ligation mixture composition: 11 μl both digested fragments, 2.5 μl ligation buffer (Fermentas), 1 μl ligase T4 (Fermentas)</li> | ||
| + | <li>Duration of ligation was about 16 hours</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <h3><div style="text-align: center;">Cloning of p53 coding sequence</div></h3> | ||
| + | <h4>Marcin</h4> | ||
| + | <p>Task 1:</p> | ||
| + | <ul> | ||
| + | <li>Transformation of chemocompetent E. coli strain DH5α</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <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>. The only modification is usage of total volume of ligation mixture prepared 03.08.09</li> | ||
| + | <li>petri dish will be held in 37°C for 16 hours</li> | ||
| + | </ul> | ||
| + | </html> | ||
Latest revision as of 07:25, 6 September 2009
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Cloning of the mgtc promoter into the pSB1A3 plasmid
Monika
Tasks
- Plasmid assembly
Methods
- The plasmid digest mix contained: 4μl purified plasmid, 2μl Tango buffer (Fermentas), 0.5μl SpeI enzyme, 0.5μl XbaI enzyme, the solution was topped up with H2O to the final volume of 20μl.
- The mgtc promoter digest mix contained: 12μl purified promoter, 3μl Tango buffer (Fermentas), 0.5μl SpeI enzyme, 0.5μl XbaI enzyme, the solution was topped up with H2O to the final volume of 30μl.
- The digest was carried out in 37°C for 3h, but 1μl CIAP enzyme was added 1h before end to mgtc promoter digest mix 1h before the end of incubation. Then enzymes were inactivated in 80°C for 20min.
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmid containing BBa_B0032 and BBa_C0040 from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Methods:
Task 2:
- 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 analyse restriction pattern of the plasmids
Results:
verification of the restriction patterns
Comment:
All isolated plasmids do not have insert. The cloning must be performed another time
Task 3:
- Another Cloning of this biobrick
Methods:
- Ligation mixture composition: 11 μl both digested fragments, 2.5 μl ligation buffer (Fermentas), 1 μl ligase T4 (Fermentas)
- Duration of ligation was about 16 hours
Cloning of p53 coding sequence
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 03.08.09
- petri dish will be held in 37°C for 16 hours
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