EPF-Lausanne/4 August 2009

Wet Lab

Miniprep of cell cultures for LovTap-TERM, LovTap, B0015 (terminator) and P1010 (death cassette).

We ran an agarose gel as a control for PCR and digestion products from the previous day: LacI-LovTap-Term E/S, LacLP-LovTap-Term E/S, T7-LovTap-Term E/S, death cass. E/S, LovTap-TERM made by PCR only (new primers), PCR neg. control, PCR pos. control, LovTap-Term E/S, death cass. E/S (again). Everything seemed to go as planned =)

As the sequencing of our first LovTap-Term might not have worked well, we did a digestion assay, cutting the LovTap-Term plasmid with PstI (this should normally give 3 fragments, since there are 2 PstI restriction sites in LovTap). --> the gel gave the expected bands!

We prepared 14 LB-Agar plates with antibiotics ampicillin and kanamycin.

Transformed DH5-alpha cells with all ligation products from the overnight reaction (16 in total): LovTap-Term (which we redid in case the first LovTap-Term isn't correct) with phosphatase treatment, LovTap-Term non-phosph.-treatment, LovTap-Term neg. control, LacI-RBS-LovTap-Term clone n°2 phosph., LacI-RBS-LovTap-Term clone n°5 phosph., LacI-RBS-LovTap-Term clone n°7 phosph., LacI-RBS-LovTap-Term clone n°2 non-phosph., LacI-RBS-LovTap-Term clone n°5 non-phosph., LacI-RBS-LovTap-Term clone n°7 non-phosph., LacI-RBS-LovTap-Term neg. control, LacI-Lov_Term (from semi-2-step PCR)- death cass. vect., LacLP-LovTap-Term-d.c. vect., T7-LovTap-Term-d.c.vect., LovTap-Term (from PCR) n°1, LovTap-Term (from PCR) n°2.

The plates were left to incubate overnight at 37°C.

People in the lab

Nath, Basile, Gab, Christian, Nicolas