Prather:Gibson CBA

Gibson Chew Back And Anneal Assembly: One Step Isothermal

Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.

Reagents

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

balance ddH2O

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

860 ul ddH2O

1.2 ml Total

Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.

Protocol

1. PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
2. Thaw assembly master mix and keep on ice until ready to be used
3. 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
4. Incubate at 50 C for 15-60 min (60 min optimal).
5. Transform cells with no more than 1 ul of assembly mixture.