Team:Paris Saclay/Notebook/August/29

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====1 - Colony PCR of Gibson assembly.====
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====1 - Clony PCR of Gibson assembly.====
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AA 1-8  Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3
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AB 1-8  Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate
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AC 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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AD 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
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AE 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
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AF 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate
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4 Colonies from purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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Pick up single clone dip in 10µL of H2O in PCR tube .
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Colonies repiquée dans 10µL d'eau.
 
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We do 8 PCR for each ligation.
 
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PCR MIX for 55 tubes:  
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Prepare PCR MIX for 55 tubes:  
* Buffer Dream Taq : 137.5µL
* Buffer Dream Taq : 137.5µL

Revision as of 15:12, 19 September 2013

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Contents

Notebook : August 29

summary

  • We got clonies on ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3

promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3t so we do Colony PCR for it

  • We didn't get clonies on Gibson so we do a gel electrophoresis of parts of Gibson assembly to verify the size
  • Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and electrophoresis of it .


lab work


  • A.aero/anaerobic regulation system


Digestion for PSB3K3



DNA 9µl
Ecoil I 2µl
PST I 2µl
H2O 5µl
buffer 2µl
total 20µl
Digestion for RBS_LacZ+Term_10(08/09/13)

B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3


DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Digestion for RBS_LacZ+Term_11(08/09/13)

B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3


DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Digestion for RBS_LacZ+Term_15(08/09/13)

B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3


DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl


Digestion for RBS_AmilCP+Term_9(02/09/13)



DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Digestion for RBS_AmilCP+Term_12(02/09/13)



DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Degestion product quantification
We used the nano drop to measure the DNA in 260nm and we found its concentration
name fnr(repressor)dig EcoilI and speI fnr nark dig EcoilI and speI
conc. 25.0ng/µl 13.6ng/µl




2 -Gel electrophoresis of Degestion product of promoter fnr(activator).

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of promoter fnr(activator)nirB clone 7 dig SPE I and PstI
  • Well 3 : 3µL of promoter fnr(activator)narG clone 6 dig SPE I and PstI
  • Well 4 : 3µL of promoter fnr(activator)nark Digested SPE I and PstI
  • Well 5: 3µL of promoter fnr(activator)narB Digested SPE I and PstI
  • Well 6: 3µL of promoter fnr(activator)narG Digested SPE I and PstI


  • Gel : 1.0%


3 -Gel electrophoresis of Degestion product of RBS_AmilCP+Term and RBS_LacZ+Term.

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of RBS_AmilCP+Term_9 digested by xpe I and PstI
  • Well 3 : 3µL of RBS_AmilCP+Term_12 digested by xpe I and PstI
  • Well 4 : 3µL of RBS_LacZ+Term_10 digested by xpe I and PstI
  • Well 5: 3µL of RBS_LacZ+Term_11 digested by xpe I and PstI
  • Well 6: 3µL of RBS_LacZ+Term_15 digested by xpe I and PstI
  • Gel : 1.0%

1 - Clony PCR of Gibson assembly.

AA 1-8 Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 AB 1-8 Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate AC 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 AD 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 AE 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate AF 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate

4 Colonies from purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3

Pick up single clone dip in 10µL of H2O in PCR tube .


Prepare PCR MIX for 55 tubes:

  • Buffer Dream Taq : 137.5µL
  • dNTP : 27.5µL
  • Oligo 44 : 27.5µL
  • Oligo 43: 27.5µL
  • Dream Taq : 11µL
  • H2O : 1.144mL


Add 2µL bacteria into 23µL PCR MIX in PCR tube PCR Program

PsPCRLacZ2108.jpg


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