Gene Synthesis by high-temperature ligation

Oligonucleotides were synthesized by Agilent and were received resuspended in 100 μL of TE buffer.

Oligonucleotides were phosphorylated using 3 μL T4 Ligase Buffer (NEB), 24 μL OLS oligonucleotides, and 3 μL T4 PNK(NEB) at 37°C for 1 hour.

The reaction was heat inactivated at 65°C for 30 min and held at 16°C.

Whole pool ligation was performed with 12 μL phosphorylated oligonucleotides, 4 μL 50% 3350 PEG (Carbowax P146–3), 2 μL 9°N buffer, 2 μL (80 U) of 9°N ligase (NEB).

Reactions were performed in a MJ Research PTC-200 thermocycler using the following program: 95°C for 2 minutes, 65°C for 24 hours, and 4°C hold.

The ligation product was used as template for gene-specific PCRs or emulsion PCR.

Gene-specific PCR was performed using 0.25 μL of the ligation product as template with gene-specific primers