Team:Paris Saclay/Notebook/August/26

From 2013.igem.org

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**''1 - Gibson assembly.''
**''1 - Gibson assembly.''
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* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
 
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* FNR_part1, FNR part1 and plasmid PSB1C3
 
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* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
 
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Sample Volume:
 
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{|
 
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| style="width:700px;border:1px solid black;vertical-align:top;" |
 
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*Tube 1 : RBS_BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
 
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*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
 
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*Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
 
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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.
Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
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We let these mix at 50°C during 1h.  
We let these mix at 50°C during 1h.  
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===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α'''====
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XiaoJing
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Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]]
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 21:28, 20 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 26

summary

  • Digestion for promoter fnr(repressor and activator) in PSB1C3 by ensyme SpeI and PstI.
  • Ligation for promoter fnr(repressor or activator) in PSB1C3 digested by ensyme SPE I and PstI and RBS_LacZ+Term_or RBS_AmilCP+Term digested by PstI and XbeI.Transformation and incubated over night.
  • 3 Gibson assembly for RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 and FNR_part1, FNR part1 and plasmid PSB1C3 and RBS_FNR part1, FNR_part2 and plasmid PSB1C3.Transformation and incubated over night.


lab work

  • A - Aerobic/Anaerobic regulation system / B - PCB sensing system
    • 1 - Gibson assembly.

Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.




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Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 20

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and PstI

XiaoJing

  • Bba_K1155000 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • Bba_K1155004 clone 6 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • Bba_K1155004 clone 7 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • Bba_K1155005 clone 6 :
    • DNA : 7µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 7µL
  • Bba_K1155005 clone 7 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • Bba_K1155006 clone 6 :
    • DNA : 4µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 10µL

Bba_K1155004 already digested by SpeI :

    • DNA : 13µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 3µL

Bba_K1155005 already digested by SpeI :

    • DNA : 10µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 6µL
  • Bba_K1155006 already digested by SpeI :
    • DNA : 9µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 7µL

We let digestions 30 minutes at 37°C.

2 - Denaturation of SpeI and PstI used for the digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

XiaoJing

Protocol : Ethanol precipitation

We used 20µL of DNA.

Nanodrop :

  • Pfnr : 91.8ng/µL
  • NarK clone 6 : 19.8ng/µL
  • Nark already digested by SpeI : 15.1ng/µL
  • NarG clone 6 : 13.4ng/µL
  • NarG clone 7 : 103.9ng/µL
  • narG already digested by SpeI : 17.4ng/µL
  • NirB clone 6 : 46ng/µL
  • NirB clone 7 : 61.6ng/µL
  • NirB already digested by SpeI : 16.1ng/µL

According to the result of the Nanodrop, we decides to do ligations with Pfnr and nirB clone 6.

3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

Used quantities :

NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pfnr with RBS-LacZ or RBS-Amil CP :

  • RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
  • Pfnr or NirB clone 6 : 5µL
  • Buffer ligase : 2µL
  • Ligase T4 : 2µL
  • H20 : 8µL

We let the ligation 1h at 37°C.

4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

Protocol : Bacterial transformation

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

Objective : obtaining FNR and BphR2

1 - Gibson assembly

XiaoJing

Tranformation of 08/21 didn't work. We will do the Gibson assembly again.

Used quantities :

  • RBS-BphR2 :
    • PSB1C3 : 3µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL ???????????????
  • FNR :
    • PSB1C3 : 3µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL ???????????????
  • RBS-FNR :
    • PSB1C3 : 3µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL ????????????

We let these mix at 50°C during 1h.

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

XiaoJing

Protocol : Bacterial transformation

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