Team:Paris Saclay/Notebook/August/1

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Revision as of 16:41, 3 October 2013

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Contents

Notebook : August 1

Lab work

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

Objective : obtaining FNR and BphR2 proteins

1 - Gel purification of PCR products : BphR2 Part I, BphR2 Part II, FNR Part I, FNR Part II, RBS-FNR Part I in plasmid pSB1C3

Xavier

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • BphR2 Part I : 44 ng/µL
  • BphR2 Part II : 64 ng/µL
  • FNR Part I : 147 ng/µL
  • FNR Part II : 140 ng/µL
  • RBS-FNR Part I : 167 ng/µL
  • PSB1C3 : 159 ng/µL

The PCR products were good. Now we will do the Gibson assembly.

2 - Gibson assembly.

Abdou, Xiaojing

Used quantities :

  • BphR2 :
    • PSB1C3 : 2µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL
    • H2O : 1µL
  • FNR :
    • PSB1C3 : 2µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL
    • H2O : 1µL
  • RBS-FNR :
    • PSB1C3 : 2µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL
    • H2O : 1µL

We let these mix at 50°C during 1h.

3 - PCR of : RBS-BphR2 Part I

Abdou

Used quantities :

    • Oligo 54F : 1µL
    • Oligo 55R : 1µL
    • Buffer phusion : 5µL
    • DNA : 0.25µL
    • dNTP : 1µL
    • Phusion : 5µL
    • H2O : 36.5µL

PCR Program :

PsPCRBSBphR23007.jpg

3 - Electrophoresis of the PCR products : RBS-BphR2 Part I

Xavier, XiaoJing

Psgel10108.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-BphR2 Part I : 197pb

We obtain fragments at the right size. We will purify it.


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