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Preparing Gibson Isothermal Assembly Mix

Purpose

The Gibson reaction can fuse multiple DNA molecules with short homology regions. This reaction is useful in general purpose cloning. This protocol describes the construction of both the buffer and enzyme master mix.

Materials

  1. 1M Tris HCl pH 7.5 (Affymetrix, 22639 1 LT)
  2. 1M MgCl₂ (Affymetrix, 78641 100 ML)
  3. Polyethalene glycol (PEG) MW 8000 (Sigma P2139)
  4. NAD Monohydrate (RPI, N30110-1.0)
  5. Phusion Polymerase (lab stock)
  6. 10mM dNTP mix (lab stock)
  7. 1M DTT (RPI, N30110-1.0)
  8. Nuclease free water (RPI, 248795)
  9. Taq DNA Ligase (lab stock)
  10. T5 exonuclease 10U/μL (NEB M0363S)
  11. PCR strip tubes with individually attached caps, 4°C
  12. 15mL Falcon Tubes
  13. 0.75mL microcentrifuge tubes, 4°C
  14. 1.7mL microcentrifuge tubes, 4°C

Procedure

Making 5x Gibson Assembly Buffer

  • Spin tubes down below after vortexing as necessary.
  • In a 15mL Falcon tube, mix
  • 3mL 1M Tris HCl, pH 7.5
  • 300μL 1M MgCl2
  • 1.5g PEG-8000
  • Vortex vigorously and leave in a 37°C shaker for 1 hour. The PEG will dissolve very slowly.
  • When fully dissolved add:
  • 20.5mg NAD (free acid)
  • 60μL 10mM dNTPs (Final concentration of dNTP will be 20µM instead of the standard 200µM since we typically have only <40bp overhangs that need to be filled in))
  • 300μL 1M DTT (154mg in 1ml of H2O, make fresh)
  • nuclease-free H₂O to 6 mL (roughly 500μL)
  • Vortex vigorously.
  • Store on ice until NAD Trihydrate fully dissolves.
  • Make 320μL aliquots in microcentrifuge tubes.
  • Transfer immediately to −80°C.
  • Store at −80°C.
  • The 5x buffer should be good for roughly a year

Making Gibson Master Mix

  • Work on ice or in a cold room.
  • In a prechilled 1.7 mL microcentrifuge tube, mix
    • 700μL H₂O
    • 320μL 5x Gibson Assembly Buffer
    • 20μL Homemade Phusion Polymerase
    • 160μL Homemade Taq DNA Ligase
    • 0.65μL 10U/μL T5 exonuclease
  • Mix well with pipette thoroughly. Do not vortex.
  • Make 15μL aliquots in prechilled PCR tubes.
  • Store at −20°C.
  • The master mix can be keep for at least a year.

Use of Gibson Master Mix

Insertion into a backbone

  • Take the 15μl aliquots out of −20°C and spin down the master mix before opening cap.
  • To 15μL of the master mix add:
  • ~100ng of the backbone
  • Equimolar insert (or equimolar each of the insert fragments)
  • Water to 20μL (do not exceed 20μL)
  • Do not add volumes < 0.5μl at any point. Dilute and/or mix DNA fragments in a separate larger volume as necessary.
  • On a thermocycler, run the following program
  • 50°C, 60 minutes
  • The incubation time can be increased for higher yields of circular plasmids.
  • For linear fragments, this time should be decreased to 15 minutes.
  • Do not gel-purify the insert fragments or the backbone as much as possible. The agarose / gel cleanup buffer tends to inhibit the isothermal assembly reaction. If you have unique and strong PCR bands, you can use your PCR product without cleaning if you are not worried about template contamination.
  • Standard nucleic acid purification kits can be used before electroporation.
  • Alternatively, as much as 2–3μL of the above reaction, without cleanup, can be tolerated in a 30μL volume of chemically-competent cells.

Reference

  • Jeff Moffitt, Cluzel Lab / Zhuang Lab @ Harvard
  • OpenWetWare Protocol