Oligo Assembly and Purification

Gibson reaction mix was produced as previously reported15 . 4mers were assembled with an equimolar 1mer ratio and an overall DNA input of 120 ng. Reactions were incubated at 50 °C for 30 min with subsequent clean up using Plasmidsafe DNAse (Epicentre). 0.5 μl of final reaction mix was used for PCR amplification with Herculase II DNA Polymerase (Agilent Technologies). PCR amplicons were digested with FastDigest SchI (Life Technologies) directly in PCR buffer for 1 h at 37 °C plus 5 min heat-inactivation at 80 °C. Subsequent 4mer purification utilised Agencourt AMPure XP (Beckman-Coulter) with PEG gradient size selection (final concentration of 7.5% PEG-8000 and 0.9 M NaCl)22 (link).