Team:Paris Saclay/Notebook/August/29

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===='''Objective : characterize Bba_K1155000 and Bba_K1155004'''====
===='''Objective : characterize Bba_K1155000 and Bba_K1155004'''====
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===='''1 - Purification of colony transformed with ligation : 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 by streaking in aerobic or anaerobic conditions'''====
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===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions'''====
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XiaoJing
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Purification of 08/28/13 didn't work. We have blue colonies for Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for nirB with RBS-LacZ-Term in PSB1C3 in anaerobic conditions. We will streak these colonies again.
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[[File:PsPfnr2908.jpg|400px]]

Revision as of 23:23, 21 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 29

summary

  • Digetion of RBS_AmilCP+Term_PSB1C3 and RBS_LacZ+Term_PSB1C3 by SpeI and PstIand check the size by Gel electrophoresis
  • check the size by Gel electrophoresis for Degestion product of promoter fnr(activator)
  • We do PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
  • we got blue color of ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.


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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Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 28

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000 and Bba_K1155004

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

Purification of 08/28/13 didn't work. We have blue colonies for Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for nirB with RBS-LacZ-Term in PSB1C3 in anaerobic conditions. We will streak these colonies again.

PsPfnr2908.jpg


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

Objective : obtaining FRN and BphR2 proteins

1 - Colony PCR of FNR, RBS-FNR and RBS-BphR2 in DH5α

XiaoJing

Transformation of 08/28/13 works. We will do a Colony PCR.

COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.

Used quantities :

  • DNA : 2µL
  • Mix : (it was divided in 8 tubes for 8 different colonies for each assembly with 23µL of mix in each tube. We do it twice.)
    • Oligo 43 : 27.5µL
    • Oligo 44 : 27.5µL
    • dNTP : 27.5µL
    • Buffer Dream Taq : 137.5µL
    • Dream Taq : 11µL
    • H2O : 1144µL

PsPCR2908.jpg

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