Team:Paris Saclay/Notebook/August/2

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===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''Objective : obtaining FNR and BphR2 proteins'''====
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XiaoJing
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===='''1 - Transformation the Gibson assembly mix (August 1st):
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====''' Transformation the Gibson assembly mix (August 1st)'''====
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plasmid pSB1C3 containing BphR2
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plasmid pSB1C3 containingFNR
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plasmid pSB1C3 containing RBS-FNR
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in DH5α'''===
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XiaoJing
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* plasmid pSB1C3 containing BphR2
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* plasmid pSB1C3 containing FNR
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* plasmid pSB1C3 contaning RBS-FNR in competent cell DH5α
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]

Latest revision as of 21:15, 4 October 2013

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Contents

Notebook : August 2

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Extraction of BBa_K1155003 from DH5α

Damir, Nadia

Tranformation from 07/30/13 works. We will extract plasmid BBa_K1155003.

Protocol : High-copy plasmid extraction

We extracted plasmid from colony 9, 11 and 12. We eluted our extracted plasmid in 50µL H2O.

2 - Electrophoresis to check the extraction of BBa_K1155003

Damir, Nadia

Psgel10208.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
  • Well 5 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • BBa_K1155003 : 2734 pb

Estimated concentrations :

  • Clone 9 : 18ng/µL
  • Clone 11 :18ng/µL
  • Clone 12 : 18ng/µL

We obtained fragments at the right size (Clone 12 starts 5 min after the others). The extraction was good. We will digest it.


Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI to check sizes of fragments

Damir, Nadia

Used quantities :

  • BBa_I732017 : 41µL
  • Buffer FD : 5µL
  • EcoRI : 2µL
  • SpeI : 2µL

We let the digestion at 1h30 at 37°C.

2 -Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI

Damir, Nadia

Psgel20208.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 et 3 : 5µL of BBa_I732017 digested by EcoRI/SpeI + 1µL 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093 bp
  • pSB1A2 : 2079 bp

We didn't obtain fragments at the right size. We will digest BBa_KI732017 again.

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

Objective : obtaining FNR and BphR2 proteins

XiaoJing

Transformation the Gibson assembly mix (August 1st)

  • plasmid pSB1C3 containing BphR2
  • plasmid pSB1C3 containing FNR
  • plasmid pSB1C3 contaning RBS-FNR in competent cell DH5α

Protocol : Bacterial transformation


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