Week 14

08/31 - 09/04

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


2. Co-transfection in yeast of nicked pRS415 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 nicked pRS415 as vector no colonies has grown on Petri dishes.


4. 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



5. 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



6. Ligation of BBa_K808007 and BBa_K808030.


7. Transformation in electro-competent DH5-α cells.


8. Preparation for sequencing.


9. Assembly of cluster 2 (BBa_K808007 + BBa_K808030).


10. PCR amplification of BBa_K936011 and BBa_K936023 using Q5 High fidelity Master Mix.