Team:Paris Saclay/Notebook/August/21

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(Created page with "{{Team:Paris_Saclay/incl_debut_generique}} ='''Notebook : August 21'''= =='''Lab work'''== ==='''A - Aerobic/Anaerobic regulation system'''=== ===='''Obtaining FNR Promoter (n...")
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Damir,Xiaojing
Damir,Xiaojing
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A1-8: FNR Promoter nirB plus  RBS_LacZ+Term_PSB1C3  
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*A1-8: FNR Promoter nirB plus  RBS_LacZ+Term_PSB1C3  
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B1-8: FNR Promoter nirB plus  RBS_Amicip+Term_PSB1C3
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*B1-8: FNR Promoter nirB plus  RBS_Amicip+Term_PSB1C3
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C1-8: FNR Promoter narG plus  RBS_LacZ+Term_PSB1C3  
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*C1-8: FNR Promoter narG plus  RBS_LacZ+Term_PSB1C3  
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D1-8: FNR Promoter narG plus  RBS_Amicip+Term_PSB1C3
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*D1-8: FNR Promoter narG plus  RBS_Amicip+Term_PSB1C3
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E1-8: FNR Promoter narK plus  RBS_LacZ+Term_PSB1C3  
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*E1-8: FNR Promoter narK plus  RBS_LacZ+Term_PSB1C3  
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F1-8: FNR Promoter narK Plus  RBS_Amicip+Term_PSB1C3
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*F1-8: FNR Promoter narK Plus  RBS_Amicip+Term_PSB1C3
*Picking of 48 colonies
*Picking of 48 colonies

Revision as of 21:02, 8 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 21

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3

1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3 for 8 colonies of each

Damir,Xiaojing

  • A1-8: FNR Promoter nirB plus RBS_LacZ+Term_PSB1C3
  • B1-8: FNR Promoter nirB plus RBS_Amicip+Term_PSB1C3
  • C1-8: FNR Promoter narG plus RBS_LacZ+Term_PSB1C3
  • D1-8: FNR Promoter narG plus RBS_Amicip+Term_PSB1C3
  • E1-8: FNR Promoter narK plus RBS_LacZ+Term_PSB1C3
  • F1-8: FNR Promoter narK Plus RBS_Amicip+Term_PSB1C3
  • Picking of 48 colonies
  • Preparation Master mix A
    • H2O : 1880µL
    • dNTP : 50µL
    • VR primer : 50µL
    • DreamTaq buffer 10x : 250µL
    • DreamTaq enzyme : 20µL
  • Preparation Master mix B
    • Master mix A :1125µL
    • VF2 primer : 25µL


Protocol : Colony PCR By using Master mix B



PCR Program :

PsPcr808.jpg

2 - Gel electrophoresis of the colony PCR products
Damir
[[]]
  • 6µL DNA Ladder
  • 10µL sample per well
  • Gel : 0.8%

Expected size : 3583bp


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

2 - Gibson assembly.

Xiaojing

  • RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
  • FNR_part1, FNR part1 and plasmid PSB1C3
  • RBS_FNR part1, FNR_part2 and plasmid PSB1C3


Sample Volume:

  • Tube 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
  • Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
  • Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)

These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.


3 - PCR pbaroduct (made the 08/01/2013) purification

Damirr available quantity:

  • FNR Part1 : 10 µl
  • FNR Part2 : 19 µl
  • RBS FNR Part1 :16.1µl
  • RBS BphR2 Part1 : 28µl
  • BphR2 Part1 : 16.4 µl
  • BphR2 Part2 : 18.9 µl

Protocol : kit purification

Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.




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