PHYSICS 102 Lecture Notes - Lecture 15: Differential Centrifugation, Restriction Enzyme, Deoxyribonuclease
Document Summary
Subcellular fractionation -> separate cellular components (bc each organelles has own nas) Velocity sedimentation: sucrose density gradient centrifuge + sendiment fractionate. Equilibrium sedimentation: centrifuge natural buoyant gradient syringe extraction. Cell fractionation: homogenate - differential centrifugation (diff speeds) fraction by size. Recall the chemical and physical properties of dna. Polyanionic -> x be hydrolysed by alkaline, neutralized by histones/na. Hydrophilic -> organic extraction (ppt) -> separate from p. More cg = higher bp -> h-bond breaks at high temp -> denature. Nucleases specifically attack + hydrolyse phosphodiester bonds in nas. Rnase is very stable -> found everywhere -> hard to purify dnase or rna. Learn the basis to nucleic acid purification techniques. Strong detergent (sds): break cell mem, denature p. Chelating agents (edta): complex divalent cation (mg2+) -> x cofactors available for nucleases. Gel: restrict mvmt of relaxed dna caused by hydrodynamic forces of mixing -> x damage.
