MBIO 4612 Lecture Notes - Lecture 1: Southern Blot, Nitrocellulose, Transfer Dna
9 Feb 2020
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Immobilize dna on permanent substrate (membrane: nylon or nitrocellulose matrix doesn"t allow diffusion of dna like a gel would, slight positive charge (binds negative dna) Restriction enzyme choice is important: don"t want to cut gene (can"t cut too frequently, only 1 copy of gene per fragment (can"t cut to infrequently, gel electrophoresis. But bands don"t tell you which has what gene need to blot: dna denaturation. Gel soaked in naoh to break h bonds: transfer dna to membrane. 2 methods for transfer: capillary, better for smaller fragments, electrophoretic, dna pulled towards positive electrode. 2 methods for immobilization: uv based cross-linking, baking (i. e. heat, making a probe. Requirements: 25-2000bp of dna/ rna, complimentary to gene of interest, labeled for detection. Steps: full/partial probes via pcr (partial = random priming, denature template via heat, add random primers (bind randomly, extend random fragments via pol.
