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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.
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(230nm) |
OD(260nm) |
OD(280nm) |
OD(260nm)/OD(230nm) |
BBa_K808030 |
0,2 |
0,674 |
0,04 |
0,00 |
0,01 |
BBa_K808031 |
0,2 |
0,560 |
0,003 |
0 |
0,01 |
- 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 |
0,8 |
0,07 |
0,017 |
0,014 |
0,24 |
1,25 |
BBa_K808007 |
1,1 |
0,133 |
0,022 |
0,017 |
0,17 |
1,28 |
- 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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