Team:Warsaw/Calendar-Main/6 May 2009
From 2009.igem.org
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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