EPF-Lausanne/26 August 2009

Wet Lab

Culture

The following bacteria have been cultivated and analyzed by the fluorescent microscope in Sebastian's lab : 3x RO2 (#4,5,10), 4x RO2-BB(#4-1/#5-1/#8-5/#10-6), 3x RO2 (#4,5,10).

Colony PCR

The result of the double transformation of yesterday gave the following number of clones :

RO2#10 - BB6 : ~ 30

RO2#8 - BB5 : ~ 30

RO2#5 - BB3 : a lot

RO2#4 - BB1 : a lot

There has been a double antibiotic resistance so there might be a good chance these bacteria have included both plasmids. To be sure of it, we did a colony PCR. We took 10 clones from both 30 clones plates and 2 clones (monoclonal) for the "a lot" plates, plus 1 "dirty take" on each plate. We used the Taq platinium protocol.

Cloning

We aimed to do the following cloning : - LacI-RBS2 -> E0240

- BB5 -> RO2

- Klenow fragment -> I13507

We first did a ""PCR"" reaction. For LacI-RBS2 and BB5, we used the Taq platinium protocl and iGEM standards. A Klenow reaction had already been done the previous day. The PCR products were then ""purified"" using the Purelink Invitrogen kit.

Digestion

Purification

Ligation

Transformation

Migration

People in the lab

Basile, Rafael, Nicolas