Team:CU Boulder/Protocols/Ligation


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Ligation

Notes:

  • Keep reaction tubes and Ligase on ice at all times
  • ng of DNA will vary depending on length ratios

Ligation set-up

  1. Mix the following
    1. Add Ligase last
    2. For help calculating DNA ratios, use the NEBioCalculator
      3. ReagentAmount
      10x Ligase Buffer2 ul
      Vector DNA (4kb)50 ng
      Insert DNA (1kb)37.5 ng
      Waterto 20 ul
      T4 Ligase1 ul
  2. Mix gently by pipetting. Spin briefly
  3. Incubate (options are for sticky ends)
    1. Option 1: incubate at 16C overnight (8-12 hours)
    2. Option 2: incubate at room temperature for 10 minutes
  4. Optional: Heat inactivate the T4 Ligase at 65C for 10 minutes
  5. After incubation, keep reaction tube on ice
  6. Transform into competent cells
Team:CU-Boulder - 2015.igem.org