Using error-prone PCR to create green fluorescent gene variants

  • Author(s) / Creator(s)
  • Many versions of green fluorescent protein (GFP) have been engineered by site-directed or random mutagenesis of native proteins. However, there are still areas where these proteins can be altered in order to provide the research community with more effective biotechnology tools. This experiment used error-prone PCR to introduce random mutations into the GFP gene in an attempt to shift the wavelength of emission or brightness of GFP. The end goal of this was to produce a laboratory exercise suitable for the undergraduate. A variety of primer pairs were used to amplify the GFP gene from pmaxGFP (Lonza). The 5' primer ended at the Met1 of the GFP gene to allow potential mutation to occur everywhere except for Met1, and annealing temperatures ranging from 47°C to 64°C were tried. However, due to time constraints and issues with primers, PCR attempts were unsuccessful at amplifying the GFP gene.

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  • Type of Item
    Research Material
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  • License
    Attribution-NonCommercial 4.0 International