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!