Team:Calgary/Notebook/Protocols/soe

From 2012.igem.org

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***Adjust annealing temperatures according to the melting temperatures of specific primers used. ***</p>
***Adjust annealing temperatures according to the melting temperatures of specific primers used. ***</p>
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<p>Following each PCR reaction, gel purification of the desired band should be carried out (using the QIAquick Gel Purification Kit or another kit). Products can subsequently be used in further PCR, or in constructions once a full circuit is assembled.</p>
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<p>Following each PCR reaction, gel purification of the desired band should be carried out (using the <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/gelextraction">QIAquick Gel Purification Kit</a> or any gel purification protocol). Products can subsequently be used in further PCR, or in constructions once a full circuit is assembled.</p>

Latest revision as of 00:09, 27 October 2012

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Splice Overlap Extension (SOE) PCR Protocol

Primers are designed against a part template sequence in order to add sequence complementary to another part that you wish to join together. In addition to standard primer design considerations, the primers should have a melting temperature of 60°C with one part, and 60°C with the other.

Reactions should be set up as follows:

Reaction Mixture

  • 5 μL 10x PCR Buffer with MgSO4
  • 5μL 10x dNTPs
  • 50 pmol each primer*
  • 50-125ng Template DNA**
  • 0.5 μL Pfu Polymerase
  • Water up to 50 μL total volume

*Primers should be added according to the desired product you want to amplify.
**During the first round of PCR, template DNA will be the part itself. Subsequent rounds of PCR will use the appropriate PCR fragments from this round of PCR which you want to build together as template. These should be added at equal concentrations, along with appropriate flanking primers. Using varying concentrations of DNA template can help to optimize the reaction.

Cycling Conditions

  • Stage 1: 95%deg;C for 2 min.
  • Stage 2: 94°C for 1 min., 55-65°C for 1 min.***, 72°C for 1min/kb
  • Stage 3: 72°C for 10 min., hold at 4°C

***Adjust annealing temperatures according to the melting temperatures of specific primers used. ***

Following each PCR reaction, gel purification of the desired band should be carried out (using the QIAquick Gel Purification Kit or any gel purification protocol). Products can subsequently be used in further PCR, or in constructions once a full circuit is assembled.