- New R0051+P0412 (I763010) ligation with a new protocol (3h at 15°C and 1.2ul Ligase).
- We transform R0051+P0412 (I763010) ligation and new S03520,S0100, B0034, J06550, S0103 biobricks.
- Lab meeting on:
- LacI cloning under the pTetR (R0040) promoter control.
- Miniprep of S03520, S0100, B0034, J06550, S0103.
- S03520, S0100, J06550, S0103 digestions with Xba/Pst1.
- B0034 digestion with Spe/Pst1.
- Run on electrophoresis gel and S03520, S0100, B0034 band extraction. We have some problems with J06550 plasmid vector due to its molecular weight (too heavy) and with S0103 insert for the same reason (too heavy). Thus, we decide to use another biobrick.
- Ligation for I763012 + J06550 (I763016), I763012 + S0103 (missed in the sendbox),B0034 + J22101 (I763015 ), B0034 + J04631 (I763020).
- We transform the ligations.
- There are colonies in the 07/30/07 plates.
- We inoculate a E0020 and R0040 colony in 5ml.
- We perform some tests to set up the fluorescence acquisition with the PMT (photomultiplier tube).
- Miniprep of :
- Digestion for:
-E0022 with Xba/Pst1 ;
-R0040 with Spe/Pst1.
- Band extraction from gel for:
- Ligations at 15°C for 3h for:
- Transformation of all ligations.
- We find colonies for all ligations, except for the first one.
- Photomultiplier adjustements
We decided to go on with our fluorescence measurements using not only the photo camera but also the photomultiplier described in the "Materials and Methods" section on our Homepage.
So, we have to adjust the PMT 814 features. First of all we calibrate this instrument by regulating the output offset to have a 0.00 output in case of photomultiplier in total darkness. Then, we adjust the PMT 814 time constant to 50 ns and its gain to 10^(-3) microA/V.
After that we have to cut the photomultiplier field of view, in order to have it the same size as the microscope's one: we do that by moving the appropiate diaphragms.
Then, we have to adjust the external voltage, a sort of gain which is useful to mantain our output signal below 10 volts as prescribed. As the maximum possible output signal, we take a field of view that contains a fluorescent bacteria population at saturation; then we make it match with a 10.00 volts output by regulating the external gain.