EPF-Lausanne/3 August 2009
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==Wet Lab== | ==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. | 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. | ||
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+ | 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) | ||
==People in the lab== | ==People in the lab== |
Revision as of 14:43, 3 August 2009
Contents |
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)
People in the lab
Nath, Basile, Gab, Christian, Nicolas