Week 14

08/31 - 09/04

1. After 72h of incubation, only contamination was observed


2. Co-transfection in yeast of nicked pRS415 nicked and Cre with extensions the protocol given by Heloise MÜLLER


3. After Four days of culture of the electroporated yeast BY4742 with Cre+ homologous extension and the plasmid pRS415 nicked as vector no colonies has grown on Petri dishes.


4. Assembly of cluster 1:


• Xba I and Pst I 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
• Spe I 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-⍺ cells.
• Preparation for sequencing.


• Assembly of cluster 2:
• PCR amplification of BBa_K936011 and BBa_K936023 using Q5 High fidelity Master Mix.