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Team:Cambridge/Notebook/7
From 2010.igem.org
Contents |
Monday
Today the whole team was in the lab at the same time - it was very exciting.
Now that we have all our oligos, Anja and Will have been assembling the lux operon with a pBAD promoter from V. fischeri using Gibson. They set a race going between the 3 different PCR machines but unfortunately they all failed for different reasons.
Paul has been working on the firefly luciferase from the registry, performing a colony PCR in order to isolate the promoter and luciferase from previous experiments to make the biobrick for submission.
Peter, Ben and Emily (who was now back from Wales) worked on the plate reader experiment that Ben and Peter had planned last week. They plated out some Top10 glycerol stocks of the promoter, rbs and registry luciferase to make liquid culture tomorrow and start the plate reader experiments on Wednesday.
Hannah and Theo also transformed the pJS555 plasmid from Jim Slock, which doesn't contain the phage DNA present in pHK555. Bill continued with his amazing python program for designing primers.
While waiting for the PCR machines to finish the second time round, we practised typing with our faces (this was typed with my nose).
Tuesday
Today Anja and Will continued working with the bacterial luciferase. The transformation had shown that the gibson had not worked, so we needed to figure out what had gone wrong.
Wednesday
After Theo spending a long time with his camera in the dark room, we have got some very cool photos of everyone on the team with bright phosphoreum plates. We also now have a lot of team photos while everyone was here because this might be the last time now until the jamboree - quite a scary prospect. We have some very cool sequence photos of people jumping off steps outside the Sedgwick museum.
Will and Anja also discovered a possible reason that their Gibson didn't work. One of the reverse primers can anneal in the wrong place with its secondary hairpin loop structure, so it seems it had annealed in the wrong place. However, they discovered that the Gibson Master Mix does work which is a relief.
Hannah and Emily continued trying to plan the experiments for when the DNA2.0 order arrives, but there is a lot to think about. Ben and Peter also carried on sorting out the plate reader experiments and should be ready to start the first one tomorrow.
Bill and Anja did something ...
Thursday
Today we had another lab meeting (finally) at 12pm. Gos, PJ and Shuna were encouraging, which was nice. They were also impressed by Bill's Gibthon program, which he continued to work on as well as helping Ben and Peter in the afternoon. Will and Anja carried on trying to make Gibson work, including designing more primers and running some more gels, while Ben and Peter made the final preparations before their plate reader experiment could start.
In the afternoon the plate reader experiment started at about 3.30pm and should run for 6 hours (all being well). Emily carried on updating the online lab book and notebook and started the experiment for transferring registry luciferase with rbs and promoter into pSB1C3 but the plasmid purification didn't work properly so will do it again tomorrow.
In other news, our order of new E-gels arrived today, which was very unexpected. So we can gel away to our hearts content!
Friday
Saturday
Sunday
