NanoBio: Ligation

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

Triple Ligation Procedure

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

Basic Double Ligation Procedure

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

CAjoF 19:18, 17 June 2008 (UTC)
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