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Team:Warsaw/Calendar-Main/28 July 2009
From 2009.igem.org
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Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid assembly
Methods:
- The plasmid digest mix was prepared as follows:
4μl purified plasmid 2μl Tango buffer (Fermentas) 1μl SpeI enzyme 1μl XbaI enzyme
The solution was topped up with H2O to the final volume of 20μl. - The mgtc promoter digest mix was prepared as follows:
10μl purified promoter 2μl Tango buffer (Fermentas) 1μl SpeI enzyme 1μl XbaI enzyme
The solution was topped up with H2O to the final volume of 20μl. - The digest was carried out in 37°C for 3h. and then inactivated in 80°C for 20min.
- The digested plasmid was separated on 1% agarose gel.
- The plasmid backbone was extracted from agarose gel using the GelOut kit (A&A Biotechnology) accrding to the manufacturers protocol (in the total volume of 50μl).
- The ligation mix was prepared as follows:
50μl purified backbone 20μl digested promoter 8μl T4 ligase buffer (Fermentas) 2μl T4 ligase (Fermentas)
- The ligation was carried out in 18°C overnight.
Construction of RBS.3-listeriolysin
Franek/Ania
Tasks:
- Transform chemocompetent cells with the ligation mixture
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