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 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP

Abdou

Protocol : Ethanol precipitation

We used 90µL of DNA. At the end, we mix our DNA with 20µL of water.

2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP

Abdou

Psgel2607.jpg
  • Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye
  • Well 2 : 6µL of DNA Ladder
  • Gel : 1%

Expected sizes :

  • RBS-AmilCP : 3500bp

We obtain fragments at the right size. We will ligate RBS-AmilCP with Term.

3 - 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

Psgel12607.jpg
  • Well 1 : 5µL of NirB+1µL of 6X loading dye
  • Well 2 : 5µL of NirB+1µL of 6X loading dye
  • 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
  • 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 : 200bp

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 :

  • NarK :
    • Buffer phusion : 10µL
    • dNTP : 1µL
    • Oligo 47 : 2µL
    • Oligo 48 : 2µL
    • Concentrated DNA : 2µL
    • Phusion : 0.25µL
  • NarG :
    • Buffer phusion : 10µL
    • dNTP : 1µL
    • Oligo 49 : 2µL
    • Oligo 50 : 2µL
    • Concentrated DNA : 2µL
    • Phusion : 0.25µL
  • NirB :
    • Buffer phusion : 10µL
    • dNTP : 1µL
    • Oligo 45 : 2µL
    • Oligo 46 : 2µL
    • Concentrated DNA : 2µL
    • Phusion : 0.25µL

PCR Program :

PsPCR2507.jpg

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

Abdou, Xavier

Psgel22607.jpg
  • 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 : 200bp

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

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

Xavier

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

At the end we mix our DNA in 20µL of H2O.

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 : Bacterial tranformation

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