PCR Overlap Extension

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{{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 ==
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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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#PCR amplify the necessary fragments separately:
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* Add end primers, then continue cycling for another 15-20 rounds.
+
##Use a proofreading polymerase enzyme.  
-
* Gel extract the correct fragment.
+
##Use an annealing temp of 60°C.
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* Clone into a T-vector, or TOPO clone.
+
#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 15:37, 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

  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.
  2. PCR amplify the necessary fragments separately:
    1. Use a proofreading polymerase enzyme.
    2. Use an annealing temp of 60°C.
  3. Clean up or gel extract the correct size band.
  4. Use cleaned up fragments as template and primers in a PCR reaction.
    1. about 1/2 to volume of the extension 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 fragment.
  7. Clone into the desired vector.
    1. Digest
    2. Ligate
    3. Transform
    4. Select
    5. Sequence
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