Team:Paris Saclay/Notebook/August/28

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We obtain fragment at the right size. The gel purification was good. We will use it for Gibson assembly.
We obtain fragment at the right size. The gel purification was good. We will use it for Gibson assembly.
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Revision as of 20:00, 28 September 2013

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Contents

Notebook : August 28

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000 and Bba_K1155004

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3.

We streak :

  • Pfnr with RBS-LacZ-Term in PSB1C3 with O2 and Xgal
  • Pfnr with RBS-Amil CP-Term in PSB1C3 with O2
  • Pfnr with RBS-Amil CP-Term in PSB1C3 without O2
  • NirB with RBS-LacZ-Term in PSB1C3 without O2 and Xgal
  • NirB with RBS-Amil CP-Term in PSB1C3 with O2
  • NirB with RBS-Amil CP-Term in PSB1C3 without O2


A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FRN and BphR2 proteins

1 - Gel purification of PSB1C3 digested by DnpI

XiaoJing

Protocol : Gel purification

Nanodrop :

  • PSB1C3 : 37.2ng/µL

2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI

XiaoJing

]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of PSB1C3 digested by DpnI+1µl of 6X loading dye
  • Gel : 1%

expected sizes :

  • PSB1C3 : 2070 bp

We obtain fragment at the right size. The gel purification was good. We will use it for Gibson assembly.

3 - Gibson assembly

XiaoJing

Used quantities :

  • RBS-BphR2 :
    • PSB1C3 : 3µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL
  • FNR :
    • PSB1C3 : 3µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL
  • RBS-FNR :
    • PSB1C3 : 3µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL

We keep these mix at 50°C for 1h.

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α

XiaoJing

Protocol : Bacterial transformation


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