SOE PCR (Splicing by Overlap-Extension)

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Eventually, a 50ul PCR.

  • 5x GC buffer (or whichever PCR buffer used): 10ul
  • Templates: Up to 100ng of largest fragment
    • Equivalent molarity for all other fragments
  • dNTPs: 1ul
  • Water:To 47.5ul
  • Phusion: 0.5ul

  • PCR for 15 cycles "using the annealing temperature of the homologous regions".
  • Extension time: use the length of the (n-1) longest fragments, where n is the total number of fragments
  • Immediately after these 15 cycles:
    • Add 1ul each of outside primers direct to the PCR tube
    • PCR for 30 cycles, using annealing temperature matching your flanking primers; extension time: use the total length of the construct

  • Run 2ul on a gel. Gel purify ending result if needed.
    • Your product will probably be a minority in a mix of DNA!