PCR Overlap Extension: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 1: | Line 1: | ||
{{back to protocols}} | {{back to protocols}} | ||
== Overview == | == Overview == | ||
Create long DNA fragments from short ones. | Create long DNA fragments from short ones. This 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. | |||
#PCR amplify the necessary fragments separately: | |||
##Use a proofreading polymerase enzyme. | |||
##Use an annealing temp of 60°C. | |||
#Clean up or gel extract the correct size band. | |||
#Use cleaned up fragments as template and primers in a PCR reaction. | |||
##about 1/2 to volume of the extension reaction should be equimolar amounts of purified fragments. | |||
##Do not use Phusion polymerase. Try Pfu Turbo. | |||
##Run 15 PCR cycles without primers. (Template extension step) | |||
#Add end primers | |||
##Continue cycling for another 15-20 rounds. | |||
#Gel extract the correct fragment. | |||
#Clone into the desired vector. | |||
##Digest | |||
##Ligate | |||
##Transform | |||
##Select | |||
##Sequence | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:DNA]] | [[Category:DNA]] | ||
[[Category:In vitro]] | [[Category:In vitro]] |
Revision as of 13:37, 14 September 2011
back to protocols | ||
Overview
Create long DNA fragments from short ones. This 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.
- PCR amplify the necessary fragments separately:
- Use a proofreading polymerase enzyme.
- Use an annealing temp of 60°C.
- Clean up or gel extract the correct size band.
- Use cleaned up fragments as template and primers in a PCR reaction.
- about 1/2 to volume of the extension reaction should be equimolar amounts of purified fragments.
- Do not use Phusion polymerase. Try Pfu Turbo.
- Run 15 PCR cycles without primers. (Template extension step)
- Add end primers
- Continue cycling for another 15-20 rounds.
- Gel extract the correct fragment.
- Clone into the desired vector.
- Digest
- Ligate
- Transform
- Select
- Sequence