Cesium Chloride Density Gradients

By: Sarah Turner

I last spoke about our BrdU project here. Once we have incubated the Antarctic fungi DNA with BrdU, we then must seperate out the unlabelled DNA from the BrdU labelled DNA. We accomplish this by ultracentrifugation in a solution of cesium chloride. Ultracentrifugation is like normal centrifugation, only it's much faster and for a lot longer. We usually spin our CsCl density gradients for 48 hours at 48,000 x gravity.

Due to the density differences of BrdU labelled DNA (BrdU labelled DNA is more dense than non-labelled DNA) the two seperate and form two bands, similarly to this:

Source

After 48 hours of ultracentrifugation, we are then able to fraction off the gradient in order to determine where the bands have developed in the tube. Fractionating separates the two bands into new tubes.

After fractionating, we precipitate the DNA out of the CsCl solution so we now have pure BrdU labelled DNA.

Here's a great video that helps to explain a similar application of CsCl density gradients, fractionating, and precipitating.

JoVE: DNA Stable-Isotope Probing (DNA-SIP)

Also, check out this video for more information on the chemistry of cesium chloride density gradients: