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Team:Warsaw/Calendar-Main/6 May 2009
From 2009.igem.org
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| + | <p><h3>Cloning of the listeriolysin gene</h3></p> | ||
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| + | <p><h4>Marcin</h4></p> | ||
| + | <br/> | ||
| + | <p>Task:</p> | ||
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| + | <li>Ligation of the listeriolysin gene to the pKS plasmid</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Methods:</p> | ||
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| + | <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> | ||
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| - | + | text="Evaluation of the DNA samples"> | |
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*Ligation of the pKS plasmid and listeriolysin gene: | *Ligation of the pKS plasmid and listeriolysin gene: | ||
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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. | 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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Revision as of 10:05, 18 July 2009
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Cloning of the listeriolysin gene
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, contain 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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