Gibson Method, aka VLIC, aka one pot isothermal assembly
Prepare a master mix, as detailed below. Store them in 15 ul aliquots at -20 °C. Then:
- Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
- Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
- Incubate at 50 °C for 15 to 60 min (60 min is optimal).
- Transform as usual