EPF-Lausanne/7 September 2009

Wet Lab

As we got our readout1 (confirmed by PCR on miniprep + digestion assay), we have done the following test : all in LB (while waiting for a better option)

-Our 3 "right" clones of readout 2, induced by trp, Atc and nothing

-Our 3 readout 1 (confirmed) induced by trp and nothing

-A control of the "evolution of fluorescence over time" using the LacI inducible RFP from the registry that we have. This, at least, won't have any interferences.

We did a PCR for inclusion of the BB (LovTAP construct) into a vector, and a purification of the PCR products. The concentrations obtained are acceptable. All were then digested with EcoRI and SpeI.

Culture

The following culture have been made in 25mL LB + corresponding antibiotic, the bacteria being taken from the glycerol stock. They will be tested at Sebastian's Lab this afternoon.

RO2: clones #4-5-10 (Amp)

RO1: clones #1-2-3 (Amp)

LacI-RFP: clones #1-2 (Chl)

After incubation at 37°C, the OD is still at 0.00. At 5:00pm, the OD is at 0.01 -> wait until tomorrow.
Finally we killed them and we will make another overnight culture to inoculate tomorrow.

RO1 + Amp (clones #1,2,3 in LB and M9+AA+thiam)

RO2 + Amp (clones #4,5,10 in LB and M9+AA+thiam)

RO1 + BB + Amp/Kana : is being transformed

RO2 + BB + Amp/Kana (clones #1,3,4 in LB and M9+AA+thiam)

LacI-RFP

Transformation

RO1 #1 + BB1

RO1 #2 + BB5

RO1 #3 + BB3

Each time we want 10 ul of DNA, but 50% massic. A little calculation that could be useful :

y = 10a / (a+b)

x = 10 - y

With : a = concentration of RO1 in ng/ul

       b = concentration of BB in ng/ul

       x = the quantity of RO1 in ul to put

       y = the quantity of BB in ul to put

Because : x + y = 10 ul, x*a = c = y*b

Ligation

The four BB (1,3,5,6) have been purified. The ligation run overnight at 4°C.

People in the lab

Mélanie, Caroline, Basile, Nicolas