PCR Overlap Extension

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(Procedure)
(Procedure)
Line 17: Line 17:
##Do not use Phusion polymerase. Try Pfu Turbo.
##Do not use Phusion polymerase. Try Pfu Turbo.
##DO not add any primers, the templates will prime eachother
##DO not add any primers, the templates will prime eachother
-
##Run 15 PCR cycles without primers.  
+
##Run 15 PCR cycles without primers.
 +
##Use an annealing temp of 60°C.  
#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction:
#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction:
##Continue cycling for another 15-20 rounds.
##Continue cycling for another 15-20 rounds.
 +
##Use an annealing temp of 72°C
#Gel extract the correct size fragment.
#Gel extract the correct size fragment.
#Clone into the desired vector.
#Clone into the desired vector.

Revision as of 16:30, 14 September 2011

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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 eachother
    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
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