Team:Paris-Saclay/Project/Notebook/Week 8

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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.
Week8-1.jpg
Checking of the purity of BBa_K098995, BBa_K274100 and pSB1A2, all already digested by DPNI
Week8-2.jpg


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.

Week8-3.jpg

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.
Week8-4.jpg


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.

Week8-5.jpg


PCR program used:

Week8-6.jpg

  • 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.


Week8-7.jpg


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.