Knockout! PCR Genotyping Experiment

Use PCR to confirm your CRISPR/Cas gene Knockout! (This is an optional follow-up experiment to the Knockout! CRISPR/Cas Lab.) After completing the Knockout! Lab, bacteria transformed with the pKO plasmid (encoding Cas9 and a guide RNA that targets the lacZ gene) should appear phenotypically white, whereas bacteria transformed with pCtrl plasmid (encoding Cas9 and a random guide RNA) should appear phenotypically blue. Use PCR to determine the genotypes of the bacteria and confirm that you successfully used the CRISPR/Cas system to disrupt the lacZ gene.  Techniques: Micropipetting, PCR, gel electrophoresis Topics: Genome editing, molecular biology, biotechnology Time required: Requires two 45-minute class periods, or a single two-hour class period  Level: Advanced high school, college