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Team:Warsaw/Calendar-Main/6 May 2009
From 2009.igem.org
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| + | <p><h3>Cloning of hly gene into pKSII+ vector</h3></p> | ||
| + | <br/> | ||
| + | <p><h4>Marcin</h4></p> | ||
| + | <br/> | ||
| + | <p>Task:</p> | ||
| + | <ul> | ||
| + | <li>Ligation of the listeriolysin gene to the pKS plasmid</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li>Evaluation of the quality of the used DNA samples:</li> | ||
| + | </ul> | ||
| + | <p>Concentration and quality of used DNA was verify via electrophoresis of DNA sample with MassRuler DNA ladder (Fermentas) and GeneRuler DNA ladder (Fermentas). 1 μl of pKS plasmid and insert were diluted to 10 μl and loaded into gel</p> | ||
| + | <br/> | ||
| + | <img src="https://static.igem.org/mediawiki/2009/8/86/Evaluation_06_05_09.png" align="center" width="35%" height="35%" | ||
| + | alt="Evaluation of the DNA samples"> | ||
| + | <br/> | ||
| + | <ul> | ||
| + | <li>Ligation of the pKS plasmid and listeriolysin gene:</li> | ||
| + | </ul> | ||
| + | <p>Proper ligation mixture composition: | ||
| + | <pre>2.5 μl insert solution, | ||
| + | 8 μl plasmid solution | ||
| + | 4 μl ligation buffer (Invitrogen, it contains PEG4500) | ||
| + | 1 μl T4 ligase (Fermentas) | ||
| + | 4.5 μl MQ water</pre></p> | ||
| + | <p>Control ligation mixture composition: the same as proper mixture except lack of insert solution</p> | ||
| + | <br/> | ||
| + | <p>Ligation was took place in room temperature during 6 hours. Ligase was subsequently inactivated by heating to 80 °C for 15 minutes. After inactivation both mixtures were frozen.</p> | ||
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Latest revision as of 15:13, 17 September 2009
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Cloning of hly gene into pKSII+ vector
Marcin
Task:
- Ligation of the listeriolysin gene to the pKS plasmid
Methods:
- Evaluation of the quality of the used DNA samples:
Concentration and quality of used DNA was verify via electrophoresis of DNA sample with MassRuler DNA ladder (Fermentas) and GeneRuler DNA ladder (Fermentas). 1 μl of pKS plasmid and insert were diluted to 10 μl and loaded into gel
- Ligation of the pKS plasmid and listeriolysin gene:
Proper ligation mixture composition:
2.5 μl insert solution, 8 μl plasmid solution 4 μl ligation buffer (Invitrogen, it contains PEG4500) 1 μl T4 ligase (Fermentas) 4.5 μl MQ water
Control ligation mixture composition: the same as proper mixture except lack of insert solution
Ligation was took place in room temperature during 6 hours. Ligase was subsequently inactivated by heating to 80 °C for 15 minutes. After inactivation both mixtures were frozen.
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