Team:Paris-Saclay/Project/Notebook/Week 12

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Revision as of 21:51, 25 September 2012

GEMOTE

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20th August

  • The 63 colonies obtained after the Gibson for the B construction have been streaked in order to isolate clones. The Petri dishes composed of LB + Ampicilline are placed at 42°C. The #11 colony is the one that turned red.
To prepare the Gibson A construction, BBa_K098995 is amplified by PCR. To verify the Gibson, a 1% Agarose gel Electrophoresis is made. We are expecting a band at a size of 975 bp. The matrix plasmid is a control.
300px


PCR program used: 300px


21th August

  • Digestion by DPNI of BBa_K098995 in order to degrade the matrix plasmid.
  • Miniprep of the BBa_K098995


22th August

PCR on the colonies obtained after the Gibson of the B construction. The 880bp of BBa_K098995 is amplified to verify that it has been well integrated in the plasmid pSB1A2. The PCR are made by pool of 5 colonies from A to I, with a positive and a negative control. A 1% Agarose gel Electrophoresis is made. We are expecting a band at 975bp. 300px


PCR program used: 300px

23rd August

Individual PCR of the 15 candidates + #11 after the PCR on colonies. This time, all the fragments of the Gibson B construction are amplified. Visualization on 0.8% Agarose gel Electrophoresis 300px

PCR program used: 300px

  • Streak of the #11 to test if it can grow at 25°C, since we saw that:
    • At 37°C, it forms small red colonies
    • At 42°C, it forms slowly small red colonies
    • At 30°C, it forms normal white colonies

24th August

  • Miniprep of the 15 candiates + #11.
  • Digestion by AseI of the 15 candidates + #11 in order to verify that each colony has the plasmid with the Gibson B construction.
  • PCR to amplify the 880bp end of BBa_K098995 enclosed in the 15 candidate colonies + #11.

PCR program used: 300px

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