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.

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.




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)
BBa_K808007	0.800	0.070	0.017	0.014	0.240	1.250
BBa_K808007	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.

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