Team:Paris Saclay/Notebook/August/27

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(→3 - Streak of NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3 and Pfnr with RBS-Amil CP-Term in PSB1C3 to purify them)
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* Well 1 : 6µL DNA ladder
* Well 1 : 6µL DNA ladder
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* Well 2 :
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* Well 3 : 20µL of pSB1C3 digested by DnpI + 4µL of 6X loading dye
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* Well 3 : 20µL of PSB1C3 digested by DnpI+4µL of 6X loading dye
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* Gel : 1%
* Gel : 1%
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Expected sizes :  
Expected sizes :  
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* PSB1C3 : 2070 bp
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* pSB1C3 : 2070 bp
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We obtain fragment at the right size for PSB1C3. We will purify it. We used an electronic eppendorf gun too fast, some product get up and fill from well 3 to well 4 and 5.
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We obtain fragment at the right size for pSB1C3. We will purify it.  
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Revision as of 15:41, 4 October 2013

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Contents

Notebook : August 27

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000 and BBa_K1155004

1 - PCR Colony of ligation Pfnr or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3

XiaoJing

Transformation of 08/26/13 works. We will do a PCR Colony.

We took a single colony and resuspend in 10µL H2O.For each Biobrick we did 6 PCR Colony.

Used quantities :

  • DNA : 2µL
  • Mix  : (it was divided in 6 tubes for each promotor with 23µL of mix in each tube)
    • Oligo 44 : 17.5µL
    • Oligo 43 : 17.5µL
    • Buffer Dream Taq : 87.5µL
    • dNTP : 17.5µL
    • Dream Taq : 7µL
    • H2O : 591µL

PCR Program :

PsPCR2708.jpg

2 - Electrophoresis of the colony PCR products : Pfnr or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

[[]]
  • Well 1 : 6µL DNA ladder
  • Well 2 to 7 : 10µL of NirB with RBS-LacZ-Term in pSB1C3 +2µL of 6X loading dye
  • Well 8 to 13 : 10µL of NirB with RBS-Amil CP-Term in pSB1C3 +2µL of 6X loading dye
  • Well 14 to 19 : 10µL of Pfnr with RBS-LacZ-Term in pSB1C3 +2µL of 6X loading dye
  • Well 20 to 25 : 10µL of Pfnr with RBS-Amil CP-Term in pSB1C3 +2µL of 6X loading dye
  • Gel : 1%

Expected size : 3583bp ????

  • 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 :

We obtain fragments at the right size for NirB with RBS-Amil CP-Term in PSB1C3 in well 12, Pfnr with RBS-LacZ-Term in pSB1C3 in well 14, 15, 18 and 19 and Pfnr with RBS-Amil CP-Term in pSB1C3 in well 20, 22 and 25. Nevertheless, electrophoresis shows that these colonies weren't pure. We will purify them by streaking.

3 - Streak colonies of NirB with RBS-Amil CP-Term in pSB1C3, Pfnr with RBS-LacZ-Term in pSB1C3 and Pfnr with RBS-Amil CP-Term in pSB1C3 to purify them

XiaoJing

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

Objective : obtaining FNR, BphR2 proteins

1 - Electrophoresis of RBS-BphR2 Part I, BphR2 Part II, FNR Part I , FNR Part II, RBS-FNR Part I and PSB1C3

XiaoJing

Gibson tranformation of the 08/27/13 didn't work. So we did an electrophoresis to check sizes and concentrations of Gibson parts.

[[]]
  • Well 1 : 6µL DNA ladder
  • Well 2 : 5µL of PSB1C3+1µL of 6X loading dye
  • Well 3 : 5µL of RBS-BphR2 Part I+1µL of 6X loading dye
  • Well 4 : 5µL of RBS-FNR Part I+1µL of 6X loading dye
  • Well 5 : 5µL of FNR Part I+1µL of 6X loading dye
  • Well 6 : 5µL of FNR Part II+1µL of 6X loading dye
  • Well 7 : 5µL of BphR2 Part II+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • PSB1C3 : 2070 bp
  • RBS-BphR2 Part I : 197 bp
  • BphR2 Part II : 790 bp
  • RBS-FNR Part I : 615 bp
  • FNR Part I : 597 bp
  • FNR Part II : 200 bp

We obtain fragment at the right size for RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I, FNR Part II but not for PSB1C3. We will do a digestion of PSB1C3 by DnpI to clean it.

2 - Digestion of pSB1C3 by DnpI

XiaoJing

Used quantities :

  • pSB1C3 : 17µL
  • Buffer : 2µL
  • DnpI : 1µL

We keep the digestion for 1h30 at 37°C.

3 - Electrophoresis of the digestion of PSB1C3 by DnpI

XiaoJing

[[]]
  • Well 1 : 6µL DNA ladder
  • Well 3 : 20µL of pSB1C3 digested by DnpI + 4µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB1C3 : 2070 bp

We obtain fragment at the right size for pSB1C3. We will purify it.


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