BIO 2133 Lecture Notes - Lecture 7: Gene Knockout, Blastocyst, Neomycin
9 Nov 2017
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Polylinkers (region full of restriction sites) allow insertion of dna fragments. Amplify your dna of interest using pcr. Digest your dna of interest by restriction enzymes - make sure to produce sticky ends. Ligation of dna: molecules with compatible ends can be joined together by dna ligase. Transformation and selection: propagation of recombinant dna, only bacteria continuing recombinant dna with the antibiotic resistance gene grow on medium with the antibiotic. Lacz in tact metabolizes lactose and turns the bacteria blue where they would stay white otherwise. Inserting the dna sequence of interest in the lacz gene disrupts its function and the recombinant bacteria will stay white. Take a single stranded dna and nd a primer that will bind to the gene of interest with a point mutation. Use pcr to make the plasmid double stranded again but with that mutation. Select for the mutant form of the dna and insert that one into the cell.
