PCR Overlap Extension: Difference between revisions
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[[Image:SOEing.PNG|400px|center]] | |||
== Overview == | == Overview == | ||
Create long DNA fragments from | Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing. | ||
== Procedure == | == Procedure == | ||
#Design Primers: | |||
##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. | |||
##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part. | |||
##The "end primers" will not have any complements and will likely only have restriction sites. | |||
#"'''Extension PCR'''" PCR amplify the necessary fragments separately | |||
##Use a proofreading polymerase enzyme. | |||
##Use an annealing temp of 60°C. | |||
#Clean up the product using a DNA column. | |||
#"'''Overlap PCR'''" Use cleaned up fragments as template in a PCR reaction: | |||
##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 add any primers; the templates will prime each-other. | |||
##Run 15 PCR cycles without primers. | |||
##Use an annealing temp of 60°C. | |||
#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction: | |||
##Continue cycling for another 15-20 rounds. | |||
##Use an annealing temp of 72°C | |||
#Gel extract the correct size fragment. | |||
#Clone into the desired vector. | |||
##Digest | |||
##Ligate | |||
##Transform | |||
##Select | |||
##Sequence | |||
==Notes== | |||
*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]] | ||
[[Category:PCR]] |
Latest revision as of 03:30, 22 December 2011
Back to protocols
Overview
Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.
Procedure
- Design Primers:
- 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.
- These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
- The "end primers" will not have any complements and will likely only have restriction sites.
- "Extension PCR" PCR amplify the necessary fragments separately
- Use a proofreading polymerase enzyme.
- Use an annealing temp of 60°C.
- Clean up the product using a DNA column.
- "Overlap PCR" Use cleaned up fragments as template in a PCR reaction:
- 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 add any primers; the templates will prime each-other.
- Run 15 PCR cycles without primers.
- Use an annealing temp of 60°C.
- "Purification PCR" Add end primers to the Overlap PCR reaction:
- Continue cycling for another 15-20 rounds.
- Use an annealing temp of 72°C
- Gel extract the correct size fragment.
- Clone into the desired vector.
- Digest
- Ligate
- Transform
- Select
- Sequence
Notes
- This protocol works best for assembling parts parts greater than 100bp. For making smaller parts see DNA Synthesis from Oligos.