Team:Paris Saclay/Notebook/August/30

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(Difference between revisions)
(Created page with "='''Notebook : August 30'''= =='''summary'''== *Digetion of RBS_AmilCP+Term_PSB1C3 and RBS_LacZ+Term_PSB1C3 by SpeI and PstIand check the size by Gel electrophoresis * check...")
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:<u>Ligation</u>
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:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
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{| border="1" align="center"
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|-
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|promoter fnr(repressor)
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|8µl
 +
|-
 +
|RBS_LacZ+Term
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|3µl
 +
|-
 +
|Ligation buffer
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|2µl
 +
|-
 +
|Ligation T4 enzyme
 +
|2µl
 +
|-
 +
|H2O
 +
|6µl
 +
|}
 +
 +
Transformation theligation and spread in LB plates with Chlorenphenicol and Xgal.
 +
incubated at 37°C in aerobic condition over night.
 +
 +
:For promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3  :
 +
 +
 +
{| border="1" align="center"
 +
|-
 +
|promoter fnr(activator)nirK
 +
|3µl
 +
|-
 +
|RBS_LacZ+Term
 +
|3µl
 +
|-
 +
|Plasmid PSB3K3
 +
|5µl
 +
|-
 +
|Ligation buffer
 +
|2µl
 +
|-
 +
|Ligation T4 enzyme
 +
|1µl
 +
|-
 +
|H2O
 +
|6µl
 +
|}
 +
 +
Transformation theligation and spread in LB plates with k and Xgal.
 +
incubated at 37°C in anaerobic condition over night.
 +
 +
 +
:For promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3  :
 +
 +
 +
{| border="1" align="center"
 +
|-
 +
|promoter fnr(repressor)
 +
|3µl
 +
|-
 +
|RBS_LacZ+Term
 +
|3µl
 +
|-
 +
|Plasmid PSB3K3
 +
|5µl
 +
|-
 +
|Ligation buffer
 +
|2µl
 +
|-
 +
|Ligation T4 enzyme
 +
|1µl
 +
|-
 +
|H2O
 +
|6µl
 +
|}
 +
 +
Transformation theligation and spread in LB plates with k and Xgal.
 +
incubated at 37°C in aerobic condition over night.

Revision as of 15:46, 19 September 2013

Contents

Notebook : August 30

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


1 -Gel electrophoresis of PCR clonies on Gibson assembly .

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well AA 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3
  • Well AB 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate3
  • Well AC 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
  • Well AD 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
  • Well AE 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
  • Well AF 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate
  • Gel : 1.0%

2 -Gel electrophoresis of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .

[[]]
  • Well 0: 6µL DNA Ladder
  • Well 1: clone1 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
  • Well 2: clone2 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
  • Well 3: clone3 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
  • Well 4: clone4 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
  • Gel : 1.0%



Ligation


For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
promoter fnr(repressor) 8µl
RBS_LacZ+Term 3µl
Ligation buffer 2µl
Ligation T4 enzyme 2µl
H2O 6µl

Transformation theligation and spread in LB plates with Chlorenphenicol and Xgal. incubated at 37°C in aerobic condition over night.

For promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3  :


promoter fnr(activator)nirK 3µl
RBS_LacZ+Term 3µl
Plasmid PSB3K3 5µl
Ligation buffer 2µl
Ligation T4 enzyme 1µl
H2O 6µl

Transformation theligation and spread in LB plates with k and Xgal. incubated at 37°C in anaerobic condition over night.


For promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3  :


promoter fnr(repressor) 3µl
RBS_LacZ+Term 3µl
Plasmid PSB3K3 5µl
Ligation buffer 2µl
Ligation T4 enzyme 1µl
H2O 6µl

Transformation theligation and spread in LB plates with k and Xgal. incubated at 37°C in aerobic condition over night.


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