Team:Warsaw/Calendar-Main/6 May 2009

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 &mu;l of pKS plasmid and insert were diluted to 10 &mu;l and loaded into gel   Ligation of the pKS plasmid and listeriolysin gene:  Proper ligation mixture composition: 2.5 &mu;l insert solution, 8 &mu;l plasmid solution 4 &mu;l ligation buffer (Invitrogen, it contains PEG4500) 1 &mu;l T4 ligase (Fermentas) 4.5 &mu;l 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 &deg;C for 15 minutes. After inactivation both mixtures were frozen.