Team:British Columbia/Protocols/Ligations


British Columbia -


(as provided by Gingko Bioworks)

  1. Thaw 10X T4 DNA Ligase Reaction Buffer (agitate to force precipitate into solution).
  2. Add 11 uL of H2O to a 200 ul PCR tube.
  3. Add 3 uL of the digested insert and 1 uL of the digested vector.
  4. Add 2 uL of 10X T4 DNA Ligase Reaction Buffer.
  5. Add 1 uL of T4 DNA Ligase enzyme (thawed).
  6. Mix the reagents by flicking the tube. Spin briefly in the microcentrifuge to collect the liquid in the bottom of the tube. The total volume in the tube should be 20 uL.
  7. Let the mix sit at room temperature for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.
  8. Use the product for transformation.