BIOL207 Lecture Notes - Lecture 26: Ribonuclease, Plasmid, Agarose Gel Electrophoresis
21 Apr 2018
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Dna is a very large negatively charged molecule. Cells are broken open by lysing or grinding in a solution that contains chemicals that protect dna while disrupting other components of the cell. Detergents: dissolve lipid membranes and denature proteins. Na+ helps to stabilise negatively charges dna and separate it from histones. A chelating agent (edta) to protect dna. Edta sequesters mg2+ ions that activate nucleases. Proteins are removed by adjusting the salt concentration, so they precipitate. The supernatant is mixed with ethanol which causes dna to precipitate. A small pellet of dna is collected by centrifugation. Dna pellet is dissolved in water with edta and ph buffer: restriction enzymes and dna methylation. Restriction enzymes naturally function as defence against viral dna in bacteria. Hydrolyse the phosphodiester bond at specific site. Sticky ends: short stretch of complementary base pairs that anneal together and aid the formation of recombinant molecules (ecori) In a genome, there are many restriction sites.
