Team:Paris Bettencourt/Protocols

Analytical digestion protocol

  • Prepare the following mix:
    • 2µL 10X Digestion buffer
    • 0.5µL Eco31I
    • 0.5µL BbsI
    • 2µL of DNA (200ng)
    • 15µL water
  • incubate 1h at 37°C

Annealing Protocol

  • Phosphorylation of the oligos
    • 5.6μL DNAse/RNAse free water
    • 6.0μL oligo 1 (10µM)
    • 6.0μL oligo 2 (10µM)
    • 2.0μL 10X T4 DNA ligase buffer
    • 0.4μL T4 PolyNucleotide Kinase
      • Total: 20μL
  • incubate 30min at 37°C
  • add 1μL of 1M NaCl
  • incube 5min at 95°C
  • let the mix cool down
  • use 2μL of the mix as a 10X solution

Chemical test for competent cells

  • 20-30 sec at 8-10 krpm for the DNA tube (from the kit).
  • Thow cell competent on ice. Label 2 ml eppendorf µtube for each concentration and put it on ice.
  • Add 1µl of DNA into each µtube.
  • Add 50µl of competent cell into each tube. Flick gently to mix. Incubate in ice for 30 minutes. Pre-heat waterbath at 42°C.
  • Heat-shock cell by placing in waterbath 1 minute. CAUTION : The top need to be close to the water level, but not immerge in.
  • Keep back the tube to ice for 5 minutes.
  • Add 200µl of SOC media, incubate 37°C for 2h. Prepare agar plate of the media that you want, and label it (3 plate for each concentration).
  • Add 20µl of each tube n the appropriate plate. Incubate 37°C for OVN (12-16h).