EPF-Lausanne/7 September 2009

  

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

Cultures
We did some cultures and put them overnight at 37°C. They were in 3 ml of medium, and tomorrow we will inoculate them in 25 ml. We used the RO1 clones 1,2,3 and put them in LB or M9, with ampicilline. We used the RO2 clones 4,5,10 and put them in LB or M9, with ampicilline. We also used the RO2 + BB clones 1,3,4 in LB or M9, with ampicilline and kana.

Ligation
The four BB (1,3,5,6) have been purified. The ligation run overnight at 4°C. 10 ng of vector for all, 6:1 molar ratio.

Gel
The host vector which is PSB4C5 was run on a gel (all the digestion product). We did a gel 1% agarose 50 ml, to purify and extract the vector only. The vector has been run on a gel. Then we did a gel extraction with a gel extraction kit.

People in the lab
Mélanie, Caroline, Basile, Nicolas

 