PCR Overlap Extension

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[[Image:SOEing.PNG|400px|center]]
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== Overview ==
== Overview ==
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Create long DNA fragments from short ones.
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Create long DNA fragments from shorter ones.  This method is also called "Splicing by Overlap Extension" or SOEing.
== Procedure ==
== Procedure ==
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* 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.
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#Design Primers:
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* Clean up or gel extract the correct size band.
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##These primers are like bridges between the two parts you want to assemble together.
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* Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
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##You will order two primers which are complements of one another.
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* Use proofreading enzyme for extension. Do not use phusion. Try Pfu Turbo.
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##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
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* Run 10-15 PCR cycles ''without'' end primers. (Template extension step)
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##The "end primers" will not have any complements and will likely only have restriction sites.
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* Add end primers, then continue cycling for another 15-20 rounds.
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#"'''Extension PCR'''" PCR amplify the necessary fragments separately
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* Gel extract the correct fragment.
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##Use a proofreading polymerase enzyme.  
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* Clone into a T-vector, or TOPO clone.
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##Use an annealing temp of 60°C.
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#Clean up the product using a DNA column.
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#"'''Overlap PCR'''" Use cleaned up fragments as template in a PCR reaction:
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##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
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##Do not use Phusion polymerase. Try Pfu Turbo.
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##Do not add any primers; the templates will prime each-other.
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##Run 15 PCR cycles without primers.
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##Use an annealing temp of 60°C.
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#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction:
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##Continue cycling for another 15-20 rounds.
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##Use an annealing temp of 72°C
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#Gel extract the correct size fragment.
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#Clone into the desired vector.
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##Digest
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##Ligate
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##Transform
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##Select
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##Sequence
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==Notes==
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*This protocol works best for assembling parts parts greater than 100bp.  For making smaller parts see [[DNA Synthesis from Oligos]].
[[Category:Protocol]]
[[Category:Protocol]]
[[Category:DNA]]
[[Category:DNA]]
[[Category:In vitro]]
[[Category:In vitro]]
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[[Category:PCR]]

Current revision

Back to protocols

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. "Extension PCR" PCR amplify the necessary fragments separately
    1. Use a proofreading polymerase enzyme.
    2. Use an annealing temp of 60°C.
  3. Clean up the product using a DNA column.
  4. "Overlap PCR" Use cleaned up fragments as template in a PCR reaction:
    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. Do not add any primers; the templates will prime each-other.
    4. Run 15 PCR cycles without primers.
    5. Use an annealing temp of 60°C.
  5. "Purification PCR" Add end primers to the Overlap PCR reaction:
    1. Continue cycling for another 15-20 rounds.
    2. Use an annealing temp of 72°C
  6. Gel extract the correct size fragment.
  7. Clone into the desired vector.
    1. Digest
    2. Ligate
    3. Transform
    4. Select
    5. Sequence

Notes

  • This protocol works best for assembling parts parts greater than 100bp. For making smaller parts see DNA Synthesis from Oligos.
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