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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.