PCR Amplification of the Green Fluorescence Protein Gene for Sub-Cloning

Royce Hogue, Eastern Washington University


We developed a plasmid cloning by PCR technique for the green fluorescence protein gene. PCR was used to amplify the GFP gene on the pGLO plasmid with custom PCR primers. The primer design included three sequences: 1) a leader sequence, 2) a restriction endonuclease recognition sequence for the cloning site, and 3) a complimentary sequence to the GFP gene. The GFP gene amplicon was ligated to pre-cut pUC18. The recombinant pUC18 was transformed using chemically competent E. coli DH5a. A blue/white screening method was used to identify the recombinant clones.