Team:Pasteur Paris/Experiments

Polymerase Chain Reaction

1) PCR Amplification using Takara Ex Taq DNA polymerase

  • In a 0.2ml tube, set up the following reaction: tableau
  • Set up the following cycles in a PCR machine
    • Initial denaturation : 94°C for 30 sec
    • 30 cycles :
      • 94°C for 30s
      • 55°C - 65°C for 1 min depending on your annealing temperature
      • 72°C 0.5-1 min per kb
    • Final extension: 72°C for 5min.

2) PCR Amplification using Phusion DNA polymerase

  • In a 0.2ml tube, set up the following reaction: tableau
  • Set up the following cycles in a PCR machine
    • Initial denaturation : 98°C for 30 sec
    • 30 cycles :
      • 94°C for 5-10s
      • 45°C - 72°C for 10 to 30s depending on your annealing temperature
      • 72°C for 15-30s per kb
    • Final extension: 72°C for 5min.

3) PCR Amplification using Q5 High Fidelity Master Mix DNA polymerase

  • In a 0.2ml tube, set up the following reaction: tableau
  • Set up the following cycles in a PCR machine
    • Initial denaturation : 98°C for 30 sec
    • 30 cycles :
      • 94°C for 5-10s
      • 45°C - 72°C for 10 to 30s depending on your annealing temperature
      • 72°C for 20-30s per kb
    • Final extension: 72°C for 2min.

Enzymatic Digestion

1) Single restriction enzyme digestion

  • In a MicroCentrifuge tube, set up the following reaction on ice: tableau
  • Pipette up and down to homogenize the solution.
  • Quick spin in a MicroCentrifuge (5s).
  • Incubation at 37°C for 1h.
  • Heat inactivation for 20min at 80°C.
    • Optional
  • Add the phosphatase: Add 1 unit of Shrimp Alkaline Phosphatase for each pmol of phosphate end.
  • Incubate for 30min at 37°C.
  • Inactivate the phosphatase at 65°C for 15 min.

2) Double digestion

  • In a MicroCentrifuge tube, set up the following reaction on ice: tableau
  • Pipette up and down to homogenize the solution.
  • Quick spin in a MicroCentrifuge (5s).
  • Incubation at 37°C for 1h.
  • Heat inactivation for 20min at 80°C.
    • Optional
  • Add the phosphatase: Add 1 unit of Shrimp Alkaline Phosphatase for each pmol of phosphate end.
  • Incubate for 30min at 37°C.
  • Inactivate the phosphatase at 65°C for 15 min.

Ligation

  • Thaw the T4 DNA Ligase Buffer and DNA at room temperature.
  • Set up the following reaction in a microcentrifuge tube on ice : tableau
  • Gently mix the reaction by pipetting up and down
  • Quick spin in a MicroCentrifuge (5s).
  • For cohesive ends, incubation at 16°C for 1h.
  • Heat inactivation for 10min at 65°C.

MiniPrep

1) Bacterial Culture Growth

  • Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic.
  • Incubate for 12–16 h at 37°C with vigorous shaking.
  • Centrifugation at > 8000 rpm (6800 x g) in MicroCentrifuge for 3 min at room temperature (15–25°C).
  • Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

2) Plasmid purification

  • Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro- centrifuge tube.
  • Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
  • Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
  • Centrifuge for 10 min at 13,000 rpm in a MicroCentrifuge.
  • Apply the supernatants to the QIAprep spin column by decanting or pipetting.
  • Centrifuge for 30–60 s. Discard the flow-through.
  • Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
  • Add 0.75 ml Buffer PE and centrifuge for 30–60 s.
  • Discard the flow-through, and centrifuge at full speed for an additional 1 min.
  • Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
  • Add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column.
  • Let stand for 1 min.
  • Centrifuge for 1 min.

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