Team:Pasteur Paris/Week 14

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Week 14 08/31 - 09/04 After 72h of incubation, only contamination was observed. Co-transfection in yeast of nicked pRS415 and Cre with extensions the protocol given by Heloise MÜLLER. After Four days of culture of the electroporated yeast BY4742 with Cre+ homologous extension and the plasmid nicked pRS415 as vector no colonies has grown on Petri dishes. Assembly of cluster 1: XbaI and PstI enzymatic digestion. Gel migration on 1% agarose gel. Gel Extraction following the Macherey-Nagel protocol. DNA Concentration Dilution 1/25 DNA Concentration (ng/µl) OD (230 nm) OD (260 nm) OD (280 nm) OD (260 nm) / OD (230 nm) BBa_K808030 0.200 0.674 0.040 0.000 0.010 BBa_K808031 0.200 0.560 0.003 0.000 0.010 MidiPrep of BBa_K808030: SpeI and PstI enzymatic digest of BBa_K808007 with the phosphatase step. Gel migration on 1% agarose gel. Gel Purification following the Macherey-Nagel protocol. DNA concentration assay using a spectrophotometer. Dilution 1/25 DNA Concentration (ng/µl) OD (230 nm) OD (260 nm) OD (280 nm) OD (260 nm) / OD (230 nm) OD (260 nm) / OD (280 nm) 808007 0.800 0.070 0.017 0.014 0.240 1.250 808007 1,100 0,133 0.022 0.017 0.170 1.280 Ligation of BBa_K808007 and BBa_K808030. Transformation in electro-competent DH5-α cells. Preparation for sequencing. Assembly of cluster 2 (BBa_K808007 + BBa_K808030). PCR amplification of BBa_K936011 and BBa_K936023 using Q5 High fidelity Master Mix. ^ Page up