Difference between revisions of "Team:UCL/Interlabstudy"

Our Lab Procedures

Cloning

We used 2A assembly (standard biobrick assembly) to clone the GFP gene downstream of the promoters. We digested the backbone that included the promoter with SpeI and PstI and the GFP construct with XbaI and PstI. We then performed a ligation to assemble the parts using T4 ligase. We performed diagnostic digestions to test if the ligation step worked and sequenced the plasmids for final confirmation that the ligation. In the final step we transformed the ligated plasmids into competent DH5 alpha cells.

Growing

Three biological replicates of each construct were used to make liquid cultures. As a positive control we used the biobrick I20270 which has a GFP gene downstream of a constitutive promoter. As negative control we used the promoters with no GFP insert. We transformed both controls from the distribution kit into competent DH5alpha cells.

From glycerol stocks, we inoculated each construct into 10ml LB cultures with chloramphenicol and incubated these overnight at 37 degrees, shaking at 300rpm.

OD measurements

The microplate reader used was called Pregnenenen 3000 . To find the OD of the overnight cultures, we took 200ul of each overnight culture and pipetted them on to a clear 96 well plate. Then we performed two 10-fold serial dilutions with LB on the plate. We measured the OD600 of the cultures on the plate, shaking for 30s before measuring.

After measurement we calculated the volume of each culture that would give an OD of 0.5 when diluted into 1ml. We centrifuged the samples at 4 C at 12000rpm for 2 minutes and removed the supernatants. Then we resuspended the pellets in 1ml of TE buffer (10mM Tris-HCl, 1mM EDTA). Afterwards, we measured the OD again with TE buffer as blank.

Fluorescence Measurements

The microplate reader was the same as before. A black 96-well plate was used. We measured the fluorescence intensity of each sample in triplicates. The excitation wavelength was ... and the reading wavelength ...