EPF-Lausanne/3 August 2009

Wet Lab

PCR of LacI-RBS, LOVTAP (to do again LOVTAP-TERM plasmid in case our clone hadn't the correct sequence), 1,5 step PCR (see lab notebook for details) to produce the whole desired part (LacI-RBS-LOVTAP-TERM, LacLP-RBS-LOVTAP-TERM, T7-RBS-LOVTAP-TERM), and finally a PCR of LOVTAP-TERM (with required plasmid) was done, to ensure having the right sequence for the LOVTAP-TERM.

First, digestion of LOVTAP-TERM (EX) and LacI-RBS (ES) was done (2 hours @ 37°C) followed by their ligation .(overnight @ 4°C).

Then, digestion of LOVTAP (ES) and TERM (EX) (2 hours @ 37°C) was done followed by their ligation. (overnight @ 4°C)

Then, digestion of T7-RBS-LOVTAP-TERM (ES) & LacLP-RBS-LOVTAP-TERM (ES) & LacI-RBS-LOVTAP-TERM (ES) and death cassette (ES) was done followed by their ligation. (death cassette + digested PCR products)

Finally, digestion of LOVTAP-TERM (ES) & death cassette (ES) was done followed by their ligation.

People in the lab

Nath, Basile, Gab, Christian, Nicolas