SOE PCR (Splicing by Overlap-Extension)

SOE is a way to join together, and then amplify, overlapping regions of DNA. This works best if the fragments are PCR products, as this does involve primer amplification. Plan to be physically next to the PCR machine when the first step is finished; avoid an extended pause at 12° or 4°.

Please read this entire protocol, as there are calculations required!

1) Step 1: 50ul PCR without primers

• • 10ul of 5x GC buffer (or whichever PCR buffer used)
  • XXul of water (calculate beforehand; total before adding Phusion should be 49.5ul)
  • 1ul of dNTPs
  • Template DNA fragments: up to 100ng of largest fragment
  • Equivalent molarity for each other fragment (100ng) ÷ (length in bp of largest fragment) * (length in bp of fragment) = ng for fragment
  • 0.5ul of Phusion

2) Step 1 PCR program:

1. 1. 98° for 30 seconds
   2. 98° for 10 seconds (Denaturing temperature)
   3. Annealing Temperature for 30 seconds (calculate annealing temperature for the overlap regions between the fragments. For NewSweetJanus, use 55°)
   4. 72° (Extension temperature; Use 30 seconds per 1kb of the length of the second-longest fragment)
   5. Go to step 2, and repeat 14 times (so, 15 cycles total)
   6. 72° for 5 minutes
   7. 12° hold

3) Step 2 PCR

• • Add 1ul of 1:50 for each of the outside PCR primers; left-most_fragment_Forward primer, right-most_fragment_Reverse primer. This 1ul is added directly into the PCR tube in step 1, while it is in the machine.

4) Step 2 PCR program:

1. 1. 98° for 30 seconds
   2. 98° for 10 seconds (Denaturing temperature)
   3. Annealing Temperature for 30 seconds (calculate based on added primers)
   4. 72° (Extension temperature; Use 30 seconds per 1kb of the total length of expected construct)
   5. Go to step 2, and repeat 29 times (so, 30 cycles total)
   6. 72° for 5 minutes
   7. 12° hold

• Transform 15ul of this PCR, without cleanup, in S. pneumoniae.
• If using in another bacteria, run 2ul on a gel. Gel purify ending result if needed (and expect to do so).

Your product will probably be a minority in a mix of DNA of different lengths!