Gibson Chew Back And Anneal Assembly: One Step Isothermal
Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.
5x Isothermal Reaction Mix
- 3 ml 1 M Tris-Hcl (pH 7.5)
- 300 ul 1 M MgCl2
- 60 ul 100 mM dGTP
- 60 ul 100 mM dATP
- 60 ul 100 mM dTTP
- 60 ul 100 mM dCTP
- 300 ul 1 M DTT
- 1.5 g PEG-8000
- 300 ul 100 mM NAD
- 6 ml Total
Assembly Master Mix
- 320 ul 5X Isothermal Master Mix
- 0.64 ul 10 U/ul T5 exonuclease
- 20 ul 2 U/ul Phusion DNA Pol
- 0.16 ul 40 000 U/ul Taq DNA Ligase
- 1.2 ml Total
Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.
- PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
- Thaw assembly master mix and keep on ice until ready to be used
- Mix 15 ul of assembly mixture with 5 ul total of cleaned PCR product (PCR Cleanup Kit or Gel Extraction) keeping DNA inserts in equimolar amounts
- Incubate at 50 C for 15-60 min (60 min optimal).
- Transform cells with no more than 1 ul of assembly mixture.