EPF-Lausanne/26 August 2009

  

26 August 2009

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 (GFP)

- BB5 (LovTAP) -> RO2 (Read out 2)

- Klenow fragment (Trp Operon) -> I13507 (RFP)

We first did a PCR reaction. For LacI-RBS2 and BB5, we used the Taq platinium protocl and iGEM standards. We also did a negative control (without template). A Klenow reaction had already been done the previous day. The PCR products were then purified using the Purelink Invitrogen kit.

We then did a digestion of RFP (I13507, cut by E and X), GFP (E0240, cut by E and X), Klenow (cut by E and N), BB5 (cut by E and S), RO2#5 (cut by E and X) and LR2 (LacI-RBS #2, cut by E and S). We did one negative control for Taq PCR and one for Klenow.

We then purified the products of the digestion.

Finally we proceeded to the ligation of LovTAP BB + RO2, RFP + Trp Operon and LacI-RBS + GFP. We did a transformation of these ligations products, with plates of LB/Agar + Amp. Unfortunately, RFP+Klenow didn't grow.

Migration
We did a migration of the double transformation plates and the digestion products. The results weren't very good : there was no band for colony product. There are two possible explanations : either the plates don't have Amp + Kana, or there was a problem during PCR. We will try to redo a colony PCR tomorrow to see if indeed we have both plasmids in the bacterium.

People in the lab
Basile, Rafael, Nicolas

 