PCR Overlap Extension

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Overview

Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.

Procedure

  1. Design Primers:
    1. These primers are like bridges between the two parts you want to assemble together.
    2. You will order two primers which are complements of one another.
    3. These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
    4. The "end primers" will not have any complements and will likely only have restriction sites.
  2. PCR amplify the necessary fragments separately "Extension PCR":
    1. Use a proofreading polymerase enzyme.
    2. Use an annealing temp of 60°C.
  3. Clean up the product using a DNA column.
  4. Use cleaned up fragments as template and primers in a PCR reaction (This is the "Overlap PCR".
    1. About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
    2. Do not use Phusion polymerase. Try Pfu Turbo.
    3. Run 15 PCR cycles without primers. (Template extension step)
  5. Add end primers
    1. Continue cycling for another 15-20 rounds.
  6. Gel extract the correct size fragment.
  7. Clone into the desired vector.
    1. Digest
    2. Ligate
    3. Transform
    4. Select
    5. Sequence
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