Caesium chloride density gradient ultracentrifugation

Separation of DNA from protein and RNA can be accomplished with CsCl gradients which separate molecules by density (Sambrook et ah 1989). Density of protein is approximately 1.2 gml-1, RNA is greater than 1.8 gml-1, and DNA is about 1.5-1.7 gml-1, depending on the amount of ethidium bromide intercalated in the DNA. A good final density of CsCl solution is 1.57-1.62 gml-1, but it may need adjustment depending on the base composition of the DNA. Ethidium bromide is a potent mutagen and is moderately toxic. Researchers should wear gloves when handling the dye. To inactivate ethidium bromide, refer to Sambrook etal. (1989). One procedure that works well for fungal DNA is:

1. Prepare solutions according to Murray and Thompson (1980):

(a) Solution 1-1.0 m CsCl (density = 1.12 gml-1), 50 dim Tris-HC1 (pH 8.0), 10 mM EDTA, 200 ¡xg ethidium bromide ml-1

(b) Solution 2-6.6 m CsCl (density = 1.82 gml-1), 50 mm Tris-HC1 (pH 8.0), 10 mM EDTA, 0.1% n-lauroyl sarcosine

(c) Solution 3-4.5 m CsCl (density = 1.55 gml-1), 50 mm Tris-HC1 (pH 8.0), 10 mM EDTA, 0.1% n-lauroyl sarcosine, 100 ¡xg ethidium bromide ml-1.

2. Dissolve DNA in 0.5 volume CsCl Solution 1 (1.0 volume is the volume of the ultracentrifuge tube). If necessary, heat at 60 ± 2 °C in a water bath until pellet dissolves.

3. Add 0.5 volume of CsCl Solution 2.

4. Add CsCl solution 1, or CsCl crystals to adjust density to 1.58 ±0.01 gml-1.

5. Transfer to ultracentrifuge tubes and fill with CsCl Solution 3.

7. Collect DNA band with a hypodermic syringe under long-wave ultraviolet light (use protective eyeglasses).

8. Remove ethidium bromide by repeated extractions with water-saturated n-butanol or isoamyl alcohol until solution is no longer pink by visual check. Then, extract two more times.

9. Dialyse overnight against a large volume of TE.

10. Precipitate DNA with alcohol (95% ethanol) and vacuum-dry or lyophilize.

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