Biology Reference
In-Depth Information
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FIGURE 4.3
Construction of the markerless fluorescent protein fusion strains using the
Red system.
1. PCR amplify the chloramphenicol or kanamycin resistance gene (light grey box) flanked by
the FLP-recombinase sequences (grey striped boxes) and the desired fluorescent protein
(dark grey box). 2. Clean up, digest and ligate both PCR products. Using the flanking primers,
PCR amplify and gel purify the entire
l
1800 bp product. 3. Create a fluorescent protein
fusion by PCR amplification from the above template using primers that contain at least 50 nt
of homology to the desired insertion site on the chromosome. 4. Transform into pKD46-
induced electrocompetent cells where recombination occurs, directed by the two 50 bp
homology regions 5. Make this strain electrocompetent and transform with pCP20 to remove
antibiotic resistance using FLP-recombinase.
