Springer Lab: Isothermal Assembly
Isothermal Assembly works by combining a cocktail of exonuclease, polymerase, and ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT overlap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50C.
Isothermal assembly reactions are stored in the common -20C freezer as 15 ul aliquots (in a labeled box).
To perform isothermal assembly:
1. PCR up your fragments of choice, and gel purify.
2. Not exceeding a total volume of 5 ul, in a PCR tube, combine fragments at equal molecular ratio [e.g. amount fragment1 = 100 ng * (fragment size 1/ fragment size 2); amount fragment2 = 100 ng * (fragment size 2/ fragment size 1) ... etc.]. If required, bring to 5 ul with ddH2O. I recommend using approx. 100 ng of plasmid backbone (fragment containing antibiotic resistance).
3. Add the combined fragments (5 ul) to 1 Isothermal Assembly reaction aliquot (15 ul) and mix by pipetting (20 ul total).
4. Place rxn. at 50C for 15 -60 min.
5. (optional for chem. transform.) Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 ul of ddH2O.
6. Transform with 1 ul of assembly rxn.
Gibson et al (2009) Nature Methods 6(5):343-345.