BIOC 300D2 Lecture Notes - Lecture 7: Gc-Content, Umber, Inflection Point
8 Jul 2016
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In this lab: this technique works half the time. Gene in plasmid with target site for mutation. Denature the plasmid and anneal the primers containing the desired mutation (amplify in opposite directions). Using vent pol extend and incorporate the mutagenic primers resulting in nicked circular strand. Digest methylated, non-mutated parental dna template with dpni. Transform the circular, nicked dsdna into competent cells. After transformation, the competent cells repair the nicks in the mutated. The bacteria are then screened for the presence of the altered gene, how efficient mutagenic reaction. As you use bigger plasmids, mutagenic efficiency decreases. Site specific nucleotide substitution: 2 non-mutagenic external oligos and 2 complementary mutagenic oligos. It can be a single nucleotide mutagenesis, deletion or small insertion. Almost anything is possible, but it decreases pcr efficiency. Quantification of nucleic acids: pcr is highly sensitive quantification method. Other techniques: dot blot, southern blot (radio-active probe)
