Team:Paris Saclay/Notebook/August/27

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='''Notebook : August 5'''=
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=='''Lab work'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
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===='''Objective : characterize Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
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===='''1 - Colony PCR of ligation Pfnr or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
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XiaoJing
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{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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Transformation of 08/26/13 works. We will do a PCR Colony.
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COLONIES PIQUEES DANS 10µL d'eau par Tube !!!!!!!!!!!!!
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Used quantities :
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* DNA : 2µL
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* Mix  : (it was divided in 6 tubes for each promotor with 23µL of mix in each tube)
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** Oligo 44 : 17.5µL
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** Oligo 43 : 17.5µL
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** Buffer Dream Taq : 87.5µL
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** dNTP : 17.5µL
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** Dream Taq : 7µL
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** H2O : 591µL 
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PCR Program :
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[[File:PsPCR2708.jpg|400px]]
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{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 18:00, 21 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 27

summary

  • We got colonies after the transformation of E.coli with the ligation mix "PnirB_PSB1C3 and RBS_LacZ_Term"

"PnirB_PSB1C3 plus RBS_AmilCP+Term" "Pndh*_PSB1C3 plus RBS_LacZ+Term" "Pndh*_PSB1C3 plus RBS_AmilCP+Term" so we tested for correct ligation with PCR on colonies

  • We did not get any colonies for the Gibson assembly so we analyzed by gel electrophoresis the parts used for the Gibson assembly to verify their sizes and concentrations.
  • Digestion DnpI purification of the the PSB1C3 clean for Gibson and electrophoresis of it .


lab work


  • A.aero/anaerobic regulation system
    • 1.Colony PCR on e.coli with FNR Promoter plus RBS_LacZ+Term_PSB1C3 or RBS_AmilCP+Term_PSB1C3 for 8 colonies of each

Xiaojing

  • A1-8: promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3
  • B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
  • C1-8: promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3
  • D1-8: promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
  • Picking of 32 colonies
  • Preparation Master mix
    • H2O : 490µL
    • dNTP : 17.5µL
    • VR primer : 17.5µL
    • VF2 primer : 17.5µL
    • DreamTaq buffer 10x : 87.5µL
    • DreamTaq enzyme : 7µL

Protocol : Colony PCR PCR Program :

PsPcr808.jpg




2 - Gel electrophoresis of the colony PCR products
[[]]
  • 6µL DNA Ladder
  • 10µL sample per well
  • Gel : 0.8%

Expected size : 3583bp

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

1 -Gel electrophoresis of parts of Gibson assembly.

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 1µL of PSB1C3 Clean
  • Well 3 : 3µL of RBS_BphR2 part1+1µl of 6X loading dy
  • Well 4 : 3µL of BphR2 part2+1µl of 6X loading dy
  • Well 5: 3µL of RBS_FNR part1 +1µl of 6X loading dy
  • Well 6: 3µL of FNR part1 +1µl of 6X loading dy
  • Well 7 : 3µL of FNR part2 +1µl of 6X loading dy
  • Gel : 1.0% 
Now we do a Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson.
PSB1C3 clean for Gibson 17µl
enzyme Dnp1 1µl
buffer 2µl
total 20µl
Incubate at 37°C for 1h30mins.
2 - Gel purification of the PSB1C3 Clean for Gibson

2.1 - Migration of 20µl PSB1C3 Clean for Gibson digested by Dnp1 '

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 1µL of PSB1C3 Clean dig Dnp1(08/06/2013)
  • Well 3 : 20µL of PSB1C3 Clean dig Dnp1(08/27/2013)


  • Gel : 1.0%


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Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 5

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Colony PCR of ligation Pfnr or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

Transformation of 08/26/13 works. We will do a PCR Colony.

COLONIES PIQUEES DANS 10µL d'eau par Tube !!!!!!!!!!!!!

Used quantities :

  • DNA : 2µL
  • Mix  : (it was divided in 6 tubes for each promotor with 23µL of mix in each tube)
    • Oligo 44 : 17.5µL
    • Oligo 43 : 17.5µL
    • Buffer Dream Taq : 87.5µL
    • dNTP : 17.5µL
    • Dream Taq : 7µL
    • H2O : 591µL

PCR Program :

PsPCR2708.jpg


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