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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 [[1]]. This PCR adds the T7 promoter (or some variant thereof).

Extension PCR: Part II