Difference between revisions of "PCR Overlap Extension"

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Back to [[protocols]]
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[[Image:SOEing.PNG|400px|center]]
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== Overview ==
 
== Overview ==
Create long DNA fragments from short ones.  This is also called "Splicing by Overlap Extension" or SOEing.
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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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##These primers are like bridges between the two parts you want to assemble together.
 
##These primers are like bridges between the two parts you want to assemble together.
 
##You will order two primers which are complements of one another.
 
##You will order two primers which are complements of one another.
##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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##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
#PCR amplify the necessary fragments separately:
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##The "end primers" will not have any complements and will likely only have restriction sites.
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#"'''Extension PCR'''" PCR amplify the necessary fragments separately  
 
##Use a proofreading polymerase enzyme.  
 
##Use a proofreading polymerase enzyme.  
 
##Use an annealing temp of 60°C.
 
##Use an annealing temp of 60°C.
#Clean up or gel extract the correct size band.
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#Clean up the product using a DNA column.
#Use cleaned up fragments as template and primers in a PCR reaction.
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#"'''Overlap PCR'''" Use cleaned up fragments as template in a PCR reaction:
##about 1/2 to volume of the extension reaction should be equimolar amounts of purified fragments.
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##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
 
##Do not use Phusion polymerase. Try Pfu Turbo.
 
##Do not use Phusion polymerase. Try Pfu Turbo.
##Run 15 PCR cycles without primers. (Template extension step)
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##Do not add any primers; the templates will prime each-other.
#Add end primers
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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:
 
##Continue cycling for another 15-20 rounds.
 
##Continue cycling for another 15-20 rounds.
#Gel extract the correct fragment.
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##Use an annealing temp of 72°C
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#Gel extract the correct size fragment.
 
#Clone into the desired vector.
 
#Clone into the desired vector.
 
##Digest
 
##Digest
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##Select
 
##Select
 
##Sequence
 
##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]]

Latest revision as of 03:30, 22 December 2011

Back to protocols

SOEing.PNG

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.