Team:Paris Saclay/Notebook/August/27

From 2013.igem.org

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='''Notebook : August 27'''=
='''Notebook : August 27'''=
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=='''summary'''==
 
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*We got colonies after the transformation of E.coli with the ligation mix "PnirB_PSB1C3 and RBS_LacZ_Term" 
 
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"PnirB_PSB1C3 plus  RBS_AmilCP+Term" 
 
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"Pndh*_PSB1C3 plus  RBS_LacZ+Term"
 
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"Pndh*_PSB1C3 plus  RBS_AmilCP+Term"
 
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so we tested for correct ligation with PCR on colonies
 
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* 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.
 
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* Digestion DnpI purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
 
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<br>
 
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
 
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===='''1 -Gel electrophoresis of  parts of Gibson assembly.'''====
 
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{|
 
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| style="width:350px;border:1px solid black;" | [[]]
 
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| style="width:350px;border:1px solid black;vertical-align:top;" |
 
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*Well 1 : 6µL DNA Ladder
 
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*Well 2 : 1µL of PSB1C3 Clean
 
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*Well 3 : 3µL of RBS_BphR2 part1+1µl of 6X loading dy
 
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*Well 4 : 3µL of BphR2 part2+1µl of 6X loading dy
 
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*Well 5: 3µL of RBS_FNR part1 +1µl of 6X loading dy
 
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*Well 6: 3µL of FNR part1 +1µl of 6X loading dy
 
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*Well 7 : 3µL of FNR part2 +1µl of 6X loading dy
 
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*Gel : 1.0%
 
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|}
 
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Now we do a Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson.
 
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{| border="1" align="center"
 
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|-o
 
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|PSB1C3 clean for Gibson
 
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|17µl
 
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|-
 
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|enzyme Dnp1
 
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|1µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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: Incubate at 37°C for 1h30mins.
 
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=====2 - Gel purification of the PSB1C3 Clean for Gibson=====
 
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''2.1 - Migration of 20µl PSB1C3 Clean for Gibson digested by Dnp1 '
 
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{|
 
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| style="width:350px;border:1px solid black;" | [[]]
 
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| style="width:350px;border:1px solid black;vertical-align:top;" |
 
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*Well 1 : 6µL DNA Ladder
 
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*Well 2 : 1µL of PSB1C3 Clean dig Dnp1(08/06/2013)
 
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*Well 3 : 20µL of  PSB1C3 Clean dig Dnp1(08/27/2013)
 
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*Gel : 1.0%
 
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|}
 
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{| border="1" align="center"
 
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||<big>Previous week</big>
 
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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 
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|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</big>]]
 
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|}
 
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{{Team:Paris_Saclay/incl_fin}}
 
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{{Team:Paris_Saclay/incl_debut_generique}}
 
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='''Notebook : August 5'''=
 
=='''Lab work'''==
=='''Lab work'''==
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We obtain fragments at the right size for NirB with RBS-Amil CP-Term in PSB1C3 in well 12, Pfnr with RBS-LacZ-Term in PSB1C3 in well 14, 15, 18 and 19 and Pfnr with RBS-Amil CP-Term in PSB1C3 in well 20, 22 and 25. Nevertheless, electrophoresis shows that these colonies weren't pure.
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We obtain fragments at the right size for NirB with RBS-Amil CP-Term in PSB1C3 in well 12, Pfnr with RBS-LacZ-Term in PSB1C3 in well 14, 15, 18 and 19 and Pfnr with RBS-Amil CP-Term in PSB1C3 in well 20, 22 and 25. Nevertheless, electrophoresis shows that these colonies weren't pure. We will purify them by streaking.
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|}
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=====3 - Streak of NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3 and Pfnr with RBS-Amil CP-Term in PSB1C3 to purify them=====
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XiaoJing
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''Objective : obtaining FNR, BphR2 proteins'''====
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===='''1 - Electrophoresis of RBS-BphR2 Part I, BphR2 Part II, FNR Part I , FNR Part II, RBS-FNR Part I and 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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Gibson tranformation of the 08/27/13 didn't work. So we did an electrophoresis to check sizes and concentrations of Gibson parts.
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|}
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{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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* Well 1 : 6µL DNA ladder
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* Well 2 : 5µL of PSB1C3+1µL of 6X loading dye
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* Well 3 : 5µL of RBS-BphR2 Part I+1µL of 6X loading dye
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* Well 4 : 5µL of RBS-FNR Part I+1µL of 6X loading dye
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* Well 5 : 5µL of FNR Part I+1µL of 6X loading dye
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* Well 6 : 5µL of FNR Part II+1µL of 6X loading dye
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* Well 7 : 5µL of BphR2 Part II+1µL of 6X loading dye
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* Gel : 1%
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|}
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Expected sizes :
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* PSB1C3 : 2070 bp
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* RBS-BphR2 Part I : 197 bp
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* BphR2 Part II : 790 bp
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* RBS-FNR Part I : 615 bp
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* FNR Part I : 597 bp
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* FNR Part II : 200 bp
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{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain fragment at the right size for RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I, FNR Part II but not for PSB1C3. We will .........
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|}
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===='''2 - Digestion of PBB1C3 by DnpI'''====
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XiaoJing
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Used quantities :
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* PSB1C3 : 17µL
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* Buffer : 2µL
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* DnpI : 1µL
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We let the digestion 1h30 at 37°C.
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===='''3 - Electrophoresis of the digestion of PBB1C3 by DnpI'''====
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 +
XiaoJing
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{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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* Well 1 : 6µL DNA ladder
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* Well 2 :
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* Well 3 : 20µL of PSB1C3 digested by DnpI+4µL of 6X loading dye
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* Gel : 1%
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|}
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Expected sizes :
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* PSB1C3 : 2070 bp
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{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain fragment at the right size for PSB1C3. We will purify it. We used an electronic pipet gun too fast, some product get up and fill from well 3 to well 4 and 5.
|}
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 18:56, 21 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 27

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

2 - Gel electrophoresis of the colony PCR products : Pfnr or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

[[]]
  • Well 1 : 6µL DNA ladder
  • Well 2 to 7 : 10µL of NirB with RBS-LacZ-Term in PSB1C3+2µL of 6X loading dye
  • Well 8 to 13 : 10µL of NirB with RBS-Amil CP-Term in PSB1C3+2µL of 6X loading dye
  • Well 14 to 19 : 10µL of Pfnr with RBS-LacZ-Term in PSB1C3+2µL of 6X loading dye
  • Well 20 to 25 : 10µL of Pfnr with RBS-Amil CP-Term in PSB1C3+2µL of 6X loading dye
  • Gel : 1%

Expected size : 3583bp ????

  • NirB with RBS-LacZ-Term in PSB1C3 :
  • NirB with RBS-Amil CP-Term in PSB1C3 :
  • Pfnr with RBS-LacZ-Term in PSB1C3 :
  • Pfnr with RBS-Amil CP-Term in PSB1C3 :

We obtain fragments at the right size for NirB with RBS-Amil CP-Term in PSB1C3 in well 12, Pfnr with RBS-LacZ-Term in PSB1C3 in well 14, 15, 18 and 19 and Pfnr with RBS-Amil CP-Term in PSB1C3 in well 20, 22 and 25. Nevertheless, electrophoresis shows that these colonies weren't pure. We will purify them by streaking.

3 - Streak of NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3 and Pfnr with RBS-Amil CP-Term in PSB1C3 to purify them

XiaoJing

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

Objective : obtaining FNR, BphR2 proteins

1 - Electrophoresis of RBS-BphR2 Part I, BphR2 Part II, FNR Part I , FNR Part II, RBS-FNR Part I and PSB1C3

XiaoJing

Gibson tranformation of the 08/27/13 didn't work. So we did an electrophoresis to check sizes and concentrations of Gibson parts.

[[]]
  • Well 1 : 6µL DNA ladder
  • Well 2 : 5µL of PSB1C3+1µL of 6X loading dye
  • Well 3 : 5µL of RBS-BphR2 Part I+1µL of 6X loading dye
  • Well 4 : 5µL of RBS-FNR Part I+1µL of 6X loading dye
  • Well 5 : 5µL of FNR Part I+1µL of 6X loading dye
  • Well 6 : 5µL of FNR Part II+1µL of 6X loading dye
  • Well 7 : 5µL of BphR2 Part II+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • PSB1C3 : 2070 bp
  • RBS-BphR2 Part I : 197 bp
  • BphR2 Part II : 790 bp
  • RBS-FNR Part I : 615 bp
  • FNR Part I : 597 bp
  • FNR Part II : 200 bp

We obtain fragment at the right size for RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I, FNR Part II but not for PSB1C3. We will .........

2 - Digestion of PBB1C3 by DnpI

XiaoJing

Used quantities :

  • PSB1C3 : 17µL
  • Buffer : 2µL
  • DnpI : 1µL

We let the digestion 1h30 at 37°C.

3 - Electrophoresis of the digestion of PBB1C3 by DnpI

XiaoJing

[[]]
  • Well 1 : 6µL DNA ladder
  • Well 2 :
  • Well 3 : 20µL of PSB1C3 digested by DnpI+4µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • PSB1C3 : 2070 bp

We obtain fragment at the right size for PSB1C3. We will purify it. We used an electronic pipet gun too fast, some product get up and fill from well 3 to well 4 and 5.

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