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Molecular Pathology

DNA Purification

Cesium Chloride (CsCl) Density Gradient Centrifugation


Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 14 April 2012, last major update April 2012
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.

Definition
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● Mixes nucleic acid, CsCl and ethidium bromide, and subjects the mix to high-speed centrifugation
● Can use for specific bands of different nucleic acid types, which can be removed and further purified with salting-out to remove residual CsCl

Theory of purification
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● CsCl is highly soluble in water and has been used to isolate many different nucleic acid types, including chromosomal, plasmid and organelle (mitochondrial or plasmid) DNAs, and different RNA types (rRNA, tRNA, or mRNAs)
● Separation is based on nucleic acid weight, and is so exact that nucleic acids of the same size and sequence can be separated based on different isotopic labels (example: N14 vs. N15)
● Technique has been used since the 1950ís, and is sensitive enough to separate similarly sized DNA fragments based on differing A-T or C-G content

Technique
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● Typically, intact cells are collected by low-speed centrifugation, lysed in alkaline conditions with a detergent, protease, and RNase to solublize lipids and digest proteins and RNA
● Alternatively, RNA can be harvested by this method, either by isolating the specific RNA nucleic acid band, or by pre-digesting the DNA in the sample with DNase
● Sample is often partially purified by short, low velocity centrifugation to remove flocculent materials
● Supernatant is then loaded over a CsCl-buffer solution and centrifuged at ultra-high speeds, causing the CsCl to form a gradient into which the nucleic acids migrate until they reach a point of neutral buoyancy (the isopycnic point)
● CSCl centrifugation results in extremely pure nucleic acids
● Following ultracentrifugation, rotor is stopped slowly with brakes off to minimize possible disturbances to nucleic acid bands
● Ethidium bromide is very hydrophobic and is removed from DNA with appropriate hydrophilic solvents; usually ethanol precipitation
● Disadvantages of technique: requires ultracentrifuge, use of mutagenic/toxic ethidium bromide, long centrifugation time (24-28 hours)

Images
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Density-gradient ultracentrifugation of DNA

Videos / animation
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Formation of DNA band


Cesium Chloride Density Gradient Centrifugation - YouTube

End of Molecular Pathology > DNA Purification > Cesium Chloride (CsCl) Density Gradient Centrifugation


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