Team:Paris-Saclay/Project/Notebook/Week 13

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='''Week 13'''=
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*Miniprep of colonies #11 and #21
*Miniprep of colonies #11 and #21
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*Plasmids of colonies #11 and #21 have been send for sequencing
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*Gibson assembly for the A construction (BBa_K098995+BBa_K274100+ BBa_J61048+Plasmid pSB1A2) and for the C construction (BBa_K115017 + BBa_C0051+166bp end of BBa_K098995+BBa_K274100+ BBa_J61048+Plasmid pSB1A2). Transformation of DH5αZ1 competent cell with construction A or construction C. Petri Dishes have been placed at 37°C for 1 day (construction A) or 2 days (construction C)
*Gibson assembly for the A construction (BBa_K098995+BBa_K274100+ BBa_J61048+Plasmid pSB1A2) and for the C construction (BBa_K115017 + BBa_C0051+166bp end of BBa_K098995+BBa_K274100+ BBa_J61048+Plasmid pSB1A2). Transformation of DH5αZ1 competent cell with construction A or construction C. Petri Dishes have been placed at 37°C for 1 day (construction A) or 2 days (construction C)
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Latest revision as of 00:45, 27 September 2012

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Week 13

27th August

Visualization by electrophoresis of the amplification of 880bp end of BBa_K098995 by PCR from the 15 candidate colonies and #11. Use of a 0.8% Agarose gel. We are expecting a band at 880 bp
Week13-1.jpg
Digestion by AseI of the 15 candidate colonies and #11. Visualization by electrophoresis on a 0.8% Agarose gel
Week13-2.jpg


  • Miniprep of colonies #11 and #21
  • Plasmids of colonies #11 and #21 have been sent for sequencing


Amplification of BBa_K274100, BBa_K115017 and BBa_J61048 by PCR using the colony #11. Visualization by electrophoresis on a 1% Agarose gel for BB3 and BB4 and a 2% Agarose gel for BB2. We are expecting a band at 123 bp for BBa_K115017, 133 bp for BBa_J61048 and 3408bp for BBa_K274100.
Week13-3.jpg


PCR program used for each biobrick amplification:

Week13-4.jpg

  • Gibson assembly for the A construction (BBa_K098995+BBa_K274100+ BBa_J61048+Plasmid pSB1A2) and for the C construction (BBa_K115017 + BBa_C0051+166bp end of BBa_K098995+BBa_K274100+ BBa_J61048+Plasmid pSB1A2). Transformation of DH5αZ1 competent cell with construction A or construction C. Petri Dishes have been placed at 37°C for 1 day (construction A) or 2 days (construction C)

28th august

Digestion by AseI and NotI of the colonies #11, #21 and the linear plasmid.

  • AseI:
    • 1 restriction site in the Plasmid pSB1A2
    • 1 restriction site in the 880bp end of BBa_K098995
  • NotI:
    • 1 restriction site either side of the B construction
Visualization by electrophoresis on a 0.8% Agarose gel
Week13-5.jpg
Amplification by PCR of the 880bp end of BBa_K098995. Visualization by electrophoresis on a 0.8% Agarose gel. We are expecting a band at 880bp.
Week13-6.jpg

PCR program used:

Week13-7.jpg

  • Amplification by PCR of the 880bp end of BBa_K098995 + BBa_K115017 contained in the colonies #11 and #21. PCR program used:

Week13-8.jpg

  • Miniprep of the colony #11.
  • Gibson assembly of the construction B (BBa_K115017+880bp end of BBa_K098995+ BBa_K274100+ BBa_J61048+ pSB1A2). Transformation of competent cells DH5α with the construction.
  • Liquid culture of the colony #11 and a control colony 11811 to prepare a test of temperature range.


Digestion of the colony #11 by AseI+HindIII. Visualization by electrophoresis on a 0.8% Agarose gel.
Week13-9.jpg


29th August

PCR on colonies to verify the construction A and C obtained by Gibson assembly. Amplification of BBa_K098995 for A and BBa_C0051 for C. Visualization by electrophoresis on a 0.8% Agarose gel. We are expecting a band at 935bp for A and 750pb for C.
Week13-10.jpg


PCR program used: Week13-11.jpg


  • Miniprep of the construction A and the construction C.
  • Performing tests on a range of temperatures with the liquid culture of the colony #11 and the control 11811. On Petri Dishes LB+ agar+Ampicilline. 7 different temperatures have been tested: 25°C, 28°C, 30°C, 35°C, 37°C, 40°C and 42°C.

Week13-12.jpg


Week13-13.jpg


  • The remaining of liquid cultures has been centrifuged to see the residue.


Week13-14.jpg

30th and 31th august

  • Amplification of BBa_K274100 and BBa_J61048 of the colonies containing the construction A or the construction C. Visualization by electrophoresis on a 0.8% Agarose gel for BBa_K274100 and a 2% Agarose gel for BBa_J61048.


Week13-15.jpg


PCR program used:

Week13-16.jpg

  • Glycerol stock of the colonies #11 and #21.


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