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.