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Team:Paris-Saclay/Project/Notebook/Week 8
From 2012.igem.org
Week 8
23rd July
| Electrophoresis of the post Gibson assembly PCR on a 1% Agarose gel. We are expecting a band at 4800bp for B, and a band at 1200bp for α and β. |  |
| Checking of the purity of BBa_K098995, BBa_K274100 and pSB1A2, all already digested by DPNI |  |
24th July
- Purification by column of BBa_K098995, BBa_K274100 and pSB1A2.
- Estimation of the concentration of BBa_K098995, BBa_K274100 and pSB1A2 by Nanovue.
| Visualization of the purification of BBa_K098995, BBa_K274100 and pSB1A2 by electrophoresis on a 1% Agarose gel. We are expecting a band at 935 bp for BBa_K098995, 3385 pb for BBa_K274100 and 2079 bp for pSB1A2. |  |
25th July
- Amplification by PCR of BBa_K098995, BBa_K274100 and pSB1A2. Visualization by electrophoresis on a 1% Agarose gel. We are expecting a band at 935 bp for BBa_K098995, 3385 pb for BBa_K274100 and 2079 bp for pSB1A2.
PCR program used:
- Treatment by DPNI on BBa_K098995, BBa_K274100 and pSB1A2 in order to eliminate the plasmid matrix.
26th July
- Purification of BBa_K098995, BBa_K274100 and pSB1A2 with the gel extraction PCR clean up kit to prepare a Gibson assembly.
- Nanovue to measure the concentration of our 3 samples: BBa_K098995, BBa_K274100 and pSB1A2.
27th July
- Gibson assembly for the B construction (BBa_K115017 + 880bp end of BBa_K098995 + BBa_K274100 + BBa_J61048 + pSB1A2). A control is done with just the plasmid treated by DPNI.
- Transformation of competent cell DH5α with the B construction. Spreading out on LB+Agar+Ampicilline, and the Petri dishes are placed at 37°C.
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