Team:Paris Saclay/Notebook/July/26

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Notebook : July 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155003

1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : RBS-AmilCP

Abdou

Protocol : Ethanol precipitation

We used 90µL of DNA. On a reprit dans 20µL d'eau !!!!!!!!!!!!!!!!!!!!!!!!

2 - Ligation of RBS-AmilCP and Term-PSB1C3

Xavier

Used quantities :

  • RBS-AmilCP : 2µL
  • Term in PSB1C3 : 2.5µL
  • Buffer : 1µL
  • Ligase : 1µL
  • H2O : 3.5µL

Objective : obtaining Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Electrophoresis of PCR products : NarK, NarG, NirB

Abdou

[[]]
  • Well 1 : 5µL of NirB+1µL of 6X loading dye
  • Well 2 : 5µL of NirB+1µL of 6X loading dye LA MEME CHOSE ???????
  • Well 3 : 5µL of NarG+1µL of 6X loading dye
  • Well 4 : 5µL of NarG+1µL of 6X loading dye
  • Well 5 : 6µL DNA Ladder
  • Well 6 : 5µL of NarK+1µL of 6X loading dye NUMERO 4 ???????????????
  • Well 7 : 5µL of NarK+1µL of 6X loading dye
  • Well 8 : 5µL of NarK+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • NarK, NarG, NirB : ...

We lost all our PCR products. We will do PCR again using more concentrated DNA and oligos.

2 - PCR of NarK, NarG, NirB

Abdou, Xavier

Used quantities : cf PCR précédente !!!!!!!!!!!!! ... ADN non dilué

PCR program : cf PCR précédente !!!!!!!!! SCHEMA !!!!!!!!!!!!!!

3 - Electrophoresis of PCR products : NarK, NarG, NirB

Abdou, Xavier

[[]]
  • Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
  • Well 2 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
  • Well 3 : 5µL of NarK+3µL H2O+1µL of 6X loading dye
  • Well 4 : 5µL of NarK+3µL H2O+1µL of 6X loading dye
  • Well 5 : 6µL DNA Ladder
  • Well 6 : 5µL of NarG+3µL H2O+1µL of 6X loading dye
  • Well 7 : 5µL of NarG+3µL H2O+1µL of 6X loading dye
  • Gel : 1.2%

Expected sizes :

  • NarK, NarG, NirB : ...

We obtain fragments at the right size. We can purify it.

4 - Gel purification of PCR products : NarK, NarG, NirB

Xavier

Protocol : Gel purification

REPRIS dans 20µL !!!!!!!!

5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI

Abdou

Quantities used :

  • NarK, NarG, NirB : 10µL
  • Buffer FD : 3µL
  • EcoRI FD : 1.5µL
  • PstI FD : 1.5µL
  • H2O : 14µL

We let the digestion at 37°C during 1h30 and then at -20°C.

B - PCB sensor system

Objective : obtaining Bba_K1155002

1 - Tranformation of Bba_K1155002 in DH5α

Abdou, Xavier

Protocol : Bacteral tranformation

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