Gibson Assembly

Isothermal Assembly A single-tube isothermal assembly reaction features three different enzymatic activities that perform in the same buffer:

• The exonuclease creates single-stranded 3´ overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region).
• The proprietary DNA polymerase fills in gaps within each annealed fragment.
• The DNA ligase seals nicks in the assembled DNA.

Isothermal Assembly works by combining a cocktail of exouclease, polymerase, an 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 over lap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50°C.

To perform isothermal assembly:

1. PCR up your fragments of choice, and gel purify. Also gel purify the cut vector

2. Not exceeding a total volume of 5 µL, in a PCR tube, combine fragments at equal molecular ration [e.g. amount fragment 1 = 100 ng * (fragment size 1 / fragment size 2); amount fragment 2 = 100 ng* (fragment size 2 / fragment size 1)... etc.]. If required, bring to 5 µL with ddH2O. We recommend using approximately 100 ng of plasmid backbone.

3. Add the combined fragments (5 µL) to one Isothermal Assembly reaction aliquot (15 µL) and mix by pipetting (20 µL total).

4. Incubate samples in a thermocycler at 50°C for 60 minutes

5. (optional for chem. transformation)Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 µL of ddH2O. [or simply dialyse]

6. Transform with 10 µL of assembly mix