PCR Overlap Extension

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(Overview)
(Procedure)
Line 7: Line 7:
##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.  
+
##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.  
#PCR amplify the necessary fragments separately:
#PCR amplify the necessary fragments separately:
##Use a proofreading polymerase enzyme.  
##Use a proofreading polymerase enzyme.  

Revision as of 15:40, 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. 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 size fragment.
  7. Clone into the desired vector.
    1. Digest
    2. Ligate
    3. Transform
    4. Select
    5. Sequence
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