Team:Paris Saclay/Notebook/August/2

From 2013.igem.org

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(→1 - Extraction of Bba_K1155003 from DH5α)
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Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
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We used colony 9, 11 and 12. ????????
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We used colony 9, 11 and 12.
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Elué dans 50µL d'H2O.
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We mix our DNA in 50µL of H2O.
===='''2 - Electrophoresis to check the extraction of Bba_K1155003'''====
===='''2 - Electrophoresis to check the extraction of Bba_K1155003'''====
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===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''Objective : obtaining FNR and BphR2 proteins'''====
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===='''1 - Tranformation of BphR2, FNR and RBS-FNR in DH5α'''====
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===='''1 - Transformation of BphR2, FNR and RBS-FNR in DH5α'''====
XiaoJing
XiaoJing

Revision as of 17:30, 27 September 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 bba_K1155003

Protocol : Hight copy plamid extraction

We used colony 9, 11 and 12. We mix our DNA in 50µL of H2O.

2 - Electrophoresis to check the extraction of Bba_K1155003

Damir, Nadia

File:Ps.jpg|350px]]
  • 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 obtain fragment 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

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

[[]]
  • 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 don'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

1 - Transformation of BphR2, FNR and RBS-FNR in DH5α

XiaoJing

Protocol : Bacterial transformation


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