Ligation of plasmid and inserts

Goal: To covalently link dsDNA to form a circular plasmid.

Important 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 vector (~2 µL, ~18 pmol vector).
   • 2-10x molar excess insert dsDNA with sticky ends
   • 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.