Gene Synthesis by Annealing

Design oligos using: Gene2Oligo software (http://berry.engin.umich.edu/gene2oligo)

Mix 1 μl of each oligonucleotide (from 100 μM stocks prepared in sterile 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). 6 to 28 of oligos for each assembly

add 10 X buffer 100  mM Tris-HCl pH 8.0, 500 mM NaCl) Add water to a final volume of 700 μl.

Incubated at 96 °C on a heating block.

Immediately turn off to allow the temperature to reach 30–35 °C (between 1.5 and 2 hours).

The final DNA concentration of the full-length insert calculated as if all the oligonucleotides annealed into the correct assembly should be approx. 140 nM (equivalent to that of each individual oligonucleotide in the mix).

Annealing plasmid and insert

Mix 0.015 pmol digested vector, and 0.09 pmol pre-annealed oligonucleotide mix (1:6) Add annealing buffer (10 mM Tris-HCl, 50 mM NaCl, pH 8.0) in a final volume of 10 μl.

Incubate at 45 °C on a heating block

Immediately turned off to reduce the temperature to 30 °C (45 minutes to 1 hour).

Add 100 μl chemically competent E. coli NEB5α or Turbo.

Incubate on ice for 30 minutes

Heat shock at 42 °C for 30 seconds

Incubate on ice for 2–5 minutes

Add 950 μl SOC

Incubate at 37 °C for one hour.

Plate on LB agar supplemented with amp or Kan.