PCR Overlap Extension

Overview
Create long DNA fragments from short ones.

Procedure

 * 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.