Silver: Ligation

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Revision as of 12:07, 2 July 2010

Using NEB T4 DNA ligase:

1. Mix:
   • 50-100 ng de-phosphorylated, digested vector DNA
   • 3-10x molar excess digested insert DNA
   • 2 μL 10x T4 DNA ligase buffer
   • x μL distilled water (final volume of reaction should be 20 μL)
   • 1 μL T4 DNA ligase
2. Mix thoroughly, incubate for 30 minutes at room temperature
3. Proceed to transformation. Store excess ligation mixture at -20 °C

Using 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 (2x T4 DNA ligase buffer) or 2uL of 10x 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.