Cesium Chloride

Purity > 99.9 %

Soluble in: Water

Store at: Room Temperature

Cesium chloride is used in the preparation of density gradients for isolation of plasmids from DNA, phage and/or cellular RNA.  Cesium chloride is also used in protein crystallization in order to create a heavy atom derivative.

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Protocol for the Preparation of Bacterial DNA (Large Scale)

Materials

Cesium Chloride

CsCl with saturated isopanol or water saturated butanol

Beckman JA-20 and VTi80 rotors or similar

1) Grow 100 ml of bacterial culture until saturation is reached.  Centrifuge cells for 10 minutes at 4000 x g.

2) Resuspend the pellet in 9.5 ml TE buffer with 0.5 ml of 10 % SDS and 50 ul of 20 mg/ml proteinase K and incubate at 37 degrees Celcius for 1 hour.

3) Add 1.8 ml of 5 M NaCl and mix.  Add 1.5 ml CTAB/NaCl solution, mix and incubate 20 minutes at 65 degrees Celcius.

4) Add an equivalent volume of cholorform/isoamyl alcohol, extract and centrifuge 10 min at 600 x g at room temperature.  Transfer supernatant with a wide-bore pipet to fresh tube.

5) Add 0.6 vol isopropanol and mix gently until DNA precipitates.  Transfer precipitate with a sealed Pasteur pipet to 1 ml of 70 % ethanol and wash.  Centrifuge 5 minutes at 10,000 x g and discard supernatant.  Resuspend pellet in 4 ml TE buffer.

6) Determine the DNA concentration with a spectrophotometer and adjust to and adjust to 50  to 100 ug/ml.

7) Add 4.3 g CsCl into 4 ml TE buffer and it dissolves.  Add 200 ul of 10 mg/ml ethidium bromide.  Transfer to 4 ml centrifuge tubes, adjust volume and balance tubes with CsCl in TE buffer (1.05 g/ml).  Seal tubes and centrifuge 4 hour at 430,000 x g, 15 degrees Celcius or overnight at 250,000 x g.

8) Visualize gradient under longwave UV lamp.  Remove band with 15-G needle and 3 ml plastic syringe.

9) Remove ethidium bromide by sequential extractions with CsCl saturated with isopropanol or water saturated butanol.

10) Dialyze overnight against 2 L TE buffer to remove CsCl. If needed precipitate DNA and resuspend at desired concentration.

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