EPF-Lausanne/30 July 2009

Wet Lab

Miniprep was done in the morning to extract the DNA for the LacI, death cassette and LovTap-Term parts.

We decided to start over again for the LacI-RBS part but with different strategies and controls:

- strategy 1: insert the digested LacI into the RBS plasmid - strategy 2: insert the digested LacI and RBS into the death cassette plasmid (problem here: difficult to purify the RBS since the part is very small)

We did a PCR of LacI, GFP (for positive control) and a negative control. Then we purified the PCR products.

Digestion of:

- LacI PCR product with E/X

- RBS vector with E/X

- neg. control with E

- LacI PCR product with E/S

- LacI vector with E

- LacI vector with S

- death cassette vector with E/P

- RBS vector with X/P

- RBS vector with X

- RBS vector with P (--> there wasn't enough DNA left for this one)

(all these controls were meant to help check whether the digestions work well)

Treated RBS E/X vector and death cassette E/P vector with phosphatase, the RBS with P/X was put at 80°C for 20 min to inactivate enzymes. We then purified everything except the RBS P/X.

We ran all products on an agarose gel to see how the digestion worked.

We then went on with the ligation:

- RBS E/X (not gel-extracted) + LacI E/S - RBS E/X (gel-extracted) + LacI E/S - LacI E/S + RBS P/X + death cass. E/P - 2 neg. controls (vector only and no DNA)

We left the ligations overnight at 4°C.

People in the lab

Basile, Gab, Christian, Mélanie, Nath