PCR Overlap Extension
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Create long DNA fragments from short ones.
- PCR amplify the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temp.
- Clean up or gel extract the correct size band.
- Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
- Use proofreading enzyme for extension. Do not use phusion. Try Pfu Turbo.
- Run 10-15 PCR cycles without end primers. (Template extension step)
- Add end primers, then continue cycling for another 15-20 rounds.
- Gel extract the correct fragment.
- Clone into a T-vector, or TOPO clone.