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Team:EPF-Lausanne/Notebook/August2011
From 2011.igem.org
Notebook: August 2011
Tuesday, 02 August 2011
Lilia came on Saturday to take the plates and here are the results:
The old competents cells seem not to work very well and the Gibson assembly failed since in the plate with only backbone we have a lot of cells therefore the backbone can closes on itself.
Alessandro make the PCR (gradient PCR in which the annealing temperature increases for each sample) to amplify TetR (putting Pconst in front of it) from Repressilator plasmid to make the Gibson for the Pconst-TetR plasmid. The result is illustrated on the right.
Since we have already amplified the backbone (pSB3K1) we can make the Gibson assembly to make the Pconst-TetR plasmid.
The latest mutation PCR yielded large amounts of product. The two bands might be a little worrying, but overall this is a lot better than any of the previous trials, with the old polymerase. The stitch PCR will be ran tomorrow.
On the side, more reporter and lysis plasmids were assembled (together with a control, containing only the backbone), from the previously made gibson fragments (PCR purified on 2nd August 2011).
Wednesday, 03 August 2011
Alessandro made glicerol stocks of Vincent's plasmid (J61002-Ptet-RFP) and purified the PCR product of the day before (Pconst-TetR) to make the Gibson assembly (using the already amplified backbone of pSB3K1). The Gibson assembly for the pSB3K1-Pconst-TetR (using either equimolar ratio of backbone-insert either 2 times insert) and for Reporter-RFP/Lysis has been plated.
