Silver: Ligation

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#*2 µL tube #2 (DNA dilution buffer)
#*2 µL tube #2 (DNA dilution buffer)
#*distilled water to final volume of 10 µL total volume
#*distilled water to final volume of 10 µL total volume
-
#Add 10 µL of freshly mixed tube #1 (T4 DNA ligase buffer).
+
#Add 10 µL of freshly mixed tube #1 (2x T4 DNA ligase buffer) or 2uL of 10x T4 DNA ligase buffer
#Add 1 µL tube #3 (T4 DNA ligase).
#Add 1 µL tube #3 (T4 DNA ligase).
#Mix well, incubate for 15 min. at room temperature.
#Mix well, incubate for 15 min. at room temperature.
#Proceed to [[Silver: Bacterial_Transformation|transformation]].  Store excess ligation mixture at -20 °C.
#Proceed to [[Silver: Bacterial_Transformation|transformation]].  Store excess ligation mixture at -20 °C.

Revision as of 10:02, 30 June 2010

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 (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.
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