Team:Paris Saclay/Notebook/August/12

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
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===='''Objective : obtaining ...'''====
+
===='''Objective : characterize Bba_K1155000'''====
===='''1 - Digestion of Bba_K1155000 by SpeI/PstI, Bba_K1155007 and Bba_K1155003 by XBaI/PstI'''====
===='''1 - Digestion of Bba_K1155000 by SpeI/PstI, Bba_K1155007 and Bba_K1155003 by XBaI/PstI'''====
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''Objective : obtaining FNR and BphR2 proteins (Gibson assembly)'''====
+
===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II '''====
===='''1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II '''====

Revision as of 18:16, 16 September 2013

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Contents

Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000

1 - Digestion of Bba_K1155000 by SpeI/PstI, Bba_K1155007 and Bba_K1155003 by XBaI/PstI

Anaïs, Nadia, XiaoJing

Used quantities :

  • Bba_K1155000 :
    • Buffer FD : 5µL
    • H2O : 38µL
    • DNA : 5µL
    • SpeI FD : 1µL
    • PstI FD : 1µL
  • Bba_K1155007 :
    • Buffer FD : 5µL
    • H2O : 23µL
    • DNA : 20µL
    • XBal FD : 1µL
    • PstI FD : 1µL
  • Bba_K1155003 :
    • Buffer FD : 5µL
    • H2O : 33µL
    • DNA : 10µL
    • XBal FD : 1µL
    • PstI FD : 1µL

We let the digestion at 37°C during 10 minutes ?????????

2 - Electrophoresis to check the digestion of Bba_K1155000 by SpeI/PstI, Bba_K1155007 and Bba_K1155003 by XBalI/PstI

Nadia

File:Psdigestion12.jpg|350px]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL Bba_K1155000 digested by SpeI/PstI+1µl of 6X loading dye
  • Well 3 : 5µL Bba_K1155007 digested by XBaI/PstI+1µl of 6X loading dye
  • Well 4 : 5µL Bba_K1155003 digested by xBaI/PstI+1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Pfnr : ...
  • RBS_LacZ_Term : 3500 kb
  • RBS_AmilCP_Term : ...
  • PSB1C3 : ...

We can't see any band for Bba_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.

3 - Digestion of Bba_K1155000 by SpeI/PstI

Anaïs, Nadia

Used quantities :

  • Buffer FD : 5µL
  • H2O : 38µL
  • DNA : 5µL
  • SpeI FD : 1µL
  • PstI FD : 1µL

We let the digestion at 37°C during 10 minutes ???????


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

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II

Damir

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL RBS-BphR2 Part I+1µl of 6X loading dye
  • Well 3 : 5µL BphR2 Part II+1µl of 6X loading dye
  • Well 4 : 5µL FNR Part I+1µl of 6X loading dye
  • Well 5 : 5µL FNR Part II+1µl of 6X loading dye
  • Well 6 : 5µL RBS-FNR Part I+1µl of 6X loading dye
  • Well 4 : 5µL BphR2 Part II+1µl of 6X loading dye
  • Gel : 0.8%

Expected size

  • RBS-BphR2 Part I : 197 kb
  • BphR2 Part II : 790 kb
  • FNR Part I : 597 kb
  • FNR Part II : 200 kb
  • RBS-FNR Part I : 615 kb
  • BphR2 Part I : 178 kb

We can't see FNR Part I, FNR Part II and BphR2 Part i fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.

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