NanoBio: Ligation

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Contents

Materials

Roche Rapid DNA Ligation Kit, Fisher catalog # NC9128816, stored at -20 °C.

Notes

Keep the total mass of DNA in the ligation below 200 ng. Using > 200 ng of DNA will actually decrease the ligation efficiency.
Do NOT make a master mix of water and reagents 1-3; it will greatly decrease the efficiency of the ligation.

Triple Ligation Procedure

  1. Mix:
    • 10-50 ng de-phosphorylated,digested Biofusion or Biobrick vector(~2 µL).
    • 3x molar excess digested insert 1 DNA.
    • 3x molar excess digested insert 2 DNA.
    • 2 µL reagent #2 (DNA dilution buffer)
    • distilled water to final volume of 10 µL total volume
  2. Add 10 µL of freshly mixed reagent #1 (T4 DNA ligase buffer) and mix well.
  3. Add 1 µL reagent #3 (T4 DNA ligase).
  4. Mix well, incubate for 15 min. at room temperature.
  5. Proceed to transformation. Store excess ligation mixture at -20 °C.

Basic Double Ligation Procedure

  1. Mix:
    • 10-50 ng de-phosphorylated, digested Biofusion or Biobrick vector (~2 µL, ~18 pmol vector).
    • 2-10x molar excess digested insert DNA.
    • 2 µL reagent #2 (DNA dilution buffer)
    • distilled water to final volume of 10 µL total volume
  2. Add 10 µL of freshly mixed reagent #1 (T4 DNA ligase buffer) and mix well.
  3. Add 1 µL reagent #3 (T4 DNA ligase).
  4. Mix well, incubate for 15 min. at room temperature.
  5. Proceed to transformation. Store excess ligation mixture at -20 °C.

CAjoF 19:18, 17 June 2008 (UTC)

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