PCR Overlap Extension

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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.