Molecular markers
DNA purification
Cesium chloride (CsCl) density gradient centrifugation

Author: Rodney E. Shackelford, D.O., Ph.D. (see Authors page)

Revised: 29 May 2018, last major update April 2012

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PubMed Search: Cesium chloride[TI] OR CsCl[TI] density gradient centrifugation

Cite this page: Shackelford, R.E. Cesium chloride (CsCl) density gradient centrifugation. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapurcesium.html. Accessed October 20th, 2018.
Definition / general
  • 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
  • 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 1950s and is sensitive enough to separate similarly sized DNA fragments based on differing A-T or C-G content
Technique
  • 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 predigesting 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)
Diagrams / tables

Images hosted on other servers:

Density gradient ultracentrifugation of DNA

Videos

Cesium chloride density gradient centrifugation