Gibson Assembly

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Gibson Assembly, also known as enzymatic or chew-back assembly, is a powerful tool used for DNA cloning. It allows you to manipulate almost any fragment of DNA in any location, alleviating the need to plan projects around restriction sites.

Contents

Overview and Background Info

Gibson Assembly uses sequence homology between fragments of DNA to join them together. A Gibson Assembly reaction mixture uses 3 enzymes: 5'-->3' exonuclease, high-fidelity polymerase and ligase. Together, these enzymes chew back the 5' end of DNA fragments (allowing fragments with sequence homology to anneal), fill in any gaps created by the chew-back, and seal the nicks in the annealed fragment. While it can be used to generate linear assemblies, Gibson is most commonly used to manipulate circular plasmids, for transformation and propagation in bacteria. Fragments for Gibson Assembly must have 15-500 bp of homology. This can be done through synthesis of entire fragments, or by amplification with "overhanging" primers

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Protocol

Materials

  • DNA fragments for assembly:
    • 50-100 ng of base vector fragments
    • 2- to 5-fold molar excess of insert (smaller fragment = higher molarity needed)
    • 15-500 base pairs of homology at ends of fragments
  • Gibson Assembly Master Mix
    • 2xNEBuilder HiFi from NEB
    • 1.33xGibson Assembly Master Mix, made from components in lab (see below)

Recipe for Gibson Assembly Master Mix

5X Reaction Buffer

To a 15 mL tube, add:

  • 1.5 g PEG-8000
  • 363.1 mg Tris-HCl
  • 46.3 mg DTT
  • 19.9 mg β-NAD
  • 600 μL 10mM dNTP mixture
  • 300 μL 1M MgCl₂

Add 3-4 mL ddH₂O and vortex thoroughly. Vortex and pipette vigorously to break up PEG-8000 crystals. Use pipette to measure volume in tube, and add water to a final volume of 6 mL. Place on ice if using immediately, or store in freezer.

This reaction buffer can be stored at -20˚C for up to 6 months, or -80˚C for at least 12 months (β-NAD denaturation).
6 mL of reaction buffer 1500 x 15 μL reactions

1.33X Master Mix, yields thirty 15 μL aliquots

To a pre-chilled 1.5 mL tube, add:

  • 292.3 μL ddH₂O (note: to create 2x Master Mix, allowing for up to 10 μL DNA per reaction, use only 142.3 μL ddH₂O)
  • 120 μL 5x Reaction Buffer
  • 0.24 μL 10U/μL T5 Exonuclease
  • 7.5 μL 2U/μL Phusion DNA Polymerase
  • 45 μL 40U/μL Taq DNA Ligase OR 15 μL 40U/μL Taq DNA Ligase + 1 μL 3000 U/μL T7 DNA Ligase + 0.6 μL 100 mM ATP + 28.4 μL ddH₂O

Aliquot in 15 μL volumes. Store at -20˚C for 3-6 months.

Reaction

If using NEBuilder HiFi:

  • Add 10 μL of DNA (+ water) to 10μL of 2xNEBuilder HiFi

If using homemade Master Mix:

  • Add 5 or 10 μL of DNA (+ water, depending on indicated concentration of Master Mix) directly to tube containing 15 μL aliquot of master mix

Incubate at 50˚C for 45-75 mins (if Master Mix contains T7 DNA Ligase: incubate for additional 15-30 mins at 25˚C or room temp).
Transform 1-4 μL into competent bacteria, or store mixture at -20˚C.

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