Team:Paris Saclay/Notebook/August/21

From 2013.igem.org

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[[File:PsPCRAmilCP2108.jpg|400px]]  
[[File:PsPCRAmilCP2108.jpg|400px]]  
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=====2 - Gel electrophoresis of the colony PCR products=====
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((((((((====3 - Culture ====
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{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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*6µL DNA Ladder
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*10µL sample per well
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*Gel : 0.8%
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Expected size : 3583bp
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====3 - Culture ====
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Nguyen
Nguyen
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*strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine
*strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine
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Incubate at 37°C over night with shake 180 rpm.
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Incubate at 37°C over night with shake 180 rpm.))))))))))))))))))))
===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
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==='''A - Aerobic/Anaerobic regulation system / B - PCB se nsing system'''===
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''1 - Gibson assembloy.'''====
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Xiaojing
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* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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===='''1 - Gibson assembly.'''====
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* FNR_part1, FNR part1 and plasmid PSB1C3
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* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
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Xiaojing
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Used quantities :
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Sample Volume:
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* RBS-BphR2 :  
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{|
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** PSB1C3 : 3µL
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** BphR2 Part I : 1µL
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| style="width:700px;border:1px solid black;vertical-align:top;" |
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** BphR2 Part II : 1µL
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*Tube 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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** Gibson mix : 15µL ???????????????
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*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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*Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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These 3 mixture is incubated at 50°C for up to one hour.
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Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
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* FNR :
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** PSB1C3 : 3µL
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** FNR Part I : 1µL
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** FNR Part II : 1µL
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** Gisbon mix : 15µL ???????????????
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* RBS-FNR :
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** PSB1C3 : 3µL
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** RBS-FNR Part I : 1µL
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** FNR Part II : 1µL
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** Gibson mix : 15µL ????????????
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We let these mix at 50°C during 1h.
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====2 - Transformation of RBS-BphR2, FNR, RBS-FNR in DH5α====
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XiaoJing
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Protocol : [[Team:Paris_Saclay/Protocols/bacterial transformation|Bacterial transformation]]
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 15:45, 17 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 21

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α

Damir, Xiaojing

Colonies repiquée dans 10µL d'eau. We do 8 PCR for each ligation.

Used quantities :

  • Mix A :
    • Buffer Dream Taq : 250µL
    • dNTP : 50µL
    • Oligo 44 : 50µL
    • Dream Taq : 20µL
    • H2O : 1.88mL
      • NirB with RBS-LacZ-Term in PSB1C3 :
        • 45µL of Mix A+1µL of oligo 45+2µL of DNA
      • NirB with RBS-Amil CP-Term in PSB1C3 :
        • 45µL of Mix A+1µL of oligo 45 +2µL of DNA
      • NarG with RBS-LacZ-Term in PSB1C3 :
        • 45µL of Mix A+1µL of oligo 41+2µL of DNA
      • NarG with RBS-Amil CP-Term in PSB1C3 :
        • 45µL of Mix A+1µL of oligo 41 +2µL of DNA
      • NarK with RBS-LacZ-Term in PSB1C3 :
        • 45µL of Mix A+1µL of oligo 47+2µL of DNA
      • NarK with RBS-Amil CP-Term in PSB1C3 :
        • 45µL of Mix A+1µL of oligo 47 +2µL of DNA
  • Mix B :
    • Mix A : 1.125mL
    • Oligo 43 : 25µL
      • NirB with RBS-LacZ-Term in PSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NirB with RBS-Amil CP-Term in PSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarG with RBS-LacZ-Term in PSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarG with RBS-Amil CP-Term in PSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarK with RBS-LacZ-Term in PSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarK with RBS-Amil CP-Term in PSB1C3 :
        • 23µL of Mix B+2µL of DNA


PCR Program for NirB with RBS-LacZ-Term in PSB1C3, NarG with RBS-LacZ-Term in PSB1C3, NarK with RBS-LacZ-Term in PSB1C3 :

PsPCRLacZ2108.jpg

PCR Program for NirB with RBS-Amil CP-Term in PSB1C3, NarG with RBS-Amil CP-Term in PSB1C3, NarK with RBS-Amil CP-Term in PSB1C3 :

PsPCRAmilCP2108.jpg

((((((((====3 - Culture ==== Nguyen

  • BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol
  • strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine

Incubate at 37°C over night with shake 180 rpm.))))))))))))))))))))

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3

1 - Electrophoresis of the digestion of Bba_J04450 by EcoRI/PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 17µL of Bba_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer
  • Gel : 1%

Expected sizes :

  • PSB3K3 : 2750 bp
  • GFP : ... NON VISIBLE SUR LE GEL ???????

We obtain a fragment at the right size. We can purify it.


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

1 - 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 let these mix at 50°C during 1h.

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

XiaoJing

Protocol : Bacterial transformation

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