SOE PCR (Splicing by Overlap-Extension)

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!