Silver: Ligation

Use Roche's Rapid DNA Ligation Kit, stored at -20 °C.
Keep the total mass of DNA below 200 ng.

1. Mix:
   • 50-100 ng de-phosphorylated, digested BioBrick vector (~2 µL, ~18 pmol vector).
   • 4-10x molar excess digested insert DNA.
   • 2 µL tube #2 (DNA dilution buffer)
   • distilled water to final volume of 10 µL total volume
2. Add 10 µL of freshly mixed tube #1 (T4 DNA ligase buffer).
3. Add 1 µL tube #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.