Team:Paris Saclay/Notebook/August/28

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*We got purple clonies on ligation promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3
*We got purple clonies on ligation promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3
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so we do a purify of 4 clonies in two plate.
+
so we do a purify of 4 clonies in two plate.incubated at 37°C in aerobic and anaerobic condition over night to verify the different.
 +
*Prepare LB plates with Chlorenphenicol and Xgal .Pufify single conle of promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 and promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 to see if color will change.
* Gel extration of Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and  do Gibson assembly again .
* Gel extration of Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and  do Gibson assembly again .

Revision as of 15:58, 12 September 2013

Notebook : August 27

summary

  • We got purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3

so we do a purify of 4 clonies in two plate.incubated at 37°C in aerobic and anaerobic condition over night to verify the different.

  • Prepare LB plates with Chlorenphenicol and Xgal .Pufify single conle of promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 and promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 to see if color will change.
  • Gel extration of Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and do Gibson assembly again .


lab work


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

  • 1 - Gel extraction of the PSB1C3 Clean for Gibson.

Protocol : Gel extraction

  • 2- Migration of 3µl PSB1C3 Clean for Gibson (product gel extraction) '
[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of PSB1C3 Clean dig Dnp1 gel extraction


  • Gel : 1.0%
  • 3- product quantification.
We used the nano drop to measure the DNA in 260nm and we found its concentration
name PSB1C3 Clean dig Dnp1 gel extraction
conc. 37.2ng/µl



  • 4 - Gibson assembly.


  • RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
  • FNR_part1, FNR part1 and plasmid PSB1C3
  • RBS_FNR part1, FNR_part2 and plasmid PSB1C3


Sample Volume:

  • Tube 1 : RBS_BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
  • Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
  • Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)

These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.



A - Aerobic/Anaerobic regulation system

  • 1 - Purify single clone of promoter fnr(activator)nirB + RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.

Protocol : Purify single clone

  • 2 - Purify single clone of promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night
  • 3 - Purify single clone of promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night


  • 4 - Purify 4 single purple colony of promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night


  • 5- Purify single clone of B5 promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night


  • 6- Purify single clone of D1, D5, D6 promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night


  • 7- Purify single clone of C1, C2, C5 ,C6promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night


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