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The Making of a T7 Promoter Variant

Gene-Specific PCR

The first PCR that is used to build a full T7 promoter variant is called a "gene-specific" PCR. The primers are chosen so as to add a ribosome-binding site (rbs) upstream of the reporter gene (here RFP or Lysis) and a terminator downstream of the gene. It is in effect a typical PCR.

Extension PCR: Part I

The PCR product from the gene-specific PCR becomes the DNA template for the second PCR, which is oftentimes called the "Extension PCR". To find out more about the protocol for this PCR, click here. This PCR adds the T7 promoter (or some variant thereof).

Extension PCR: Part II

In the second phase of the Extension PCR, Gibson overhangs are added to the endpieces of the PCR product of the previous Extension PCR. These Gibson overhangs will allow for the Gibson Assembly of this promoter construct into a plasmid.