Silver: Ligation

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Current revision (11:09, 2 July 2010) (view source)
 
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Use Roche's [http://www.roche-applied-science.com/fst/amplification.htm?/sis/amplification/pifs/cloning/rapid_ligation_kit.htm Rapid DNA Ligation Kit], stored at -20 °C.  <br>
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'''Using NEB T4 DNA ligase''':
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#Mix:
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#*50-100 ng de-phosphorylated, [[Silver: Restriction_Digest|digested]] vector DNA
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#*3-10x molar excess [[Silver: Restriction_Digest|digested]] insert DNA
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#*2 μL 10x T4 DNA ligase buffer
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#*x μL distilled water (final volume of reaction should be 20 μL)
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#*1 μL T4 DNA ligase
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#Mix thoroughly, incubate for 30 minutes at room temperature
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#Proceed to [[Silver: Bacterial_Transformation|transformation]]. Store excess ligation mixture at -20 °C
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'''Using Roche's [http://www.roche-applied-science.com/fst/amplification.htm?/sis/amplification/pifs/cloning/rapid_ligation_kit.htm Rapid DNA Ligation Kit], stored at -20 °C.''' <br>
Keep the total mass of DNA below 200 ng.
Keep the total mass of DNA below 200 ng.
#Mix:  
#Mix:  
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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
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#Add 10 µL of freshly mixed tube #1 (T4 DNA ligase buffer).
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#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.

Current revision

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.
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