Preparation of Sonicated Salmon Sperm DNA¶
Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
Preparation of Sonicated Salmon Sperm DNA
- Use Pharmacia #27-4564-01. With clean flamed scissors and forceps, weigh 0.25 g/50 ml conical and add 50 ml/conical of 0.02 M Tris, pH 7.6. Allow to dissolve over several days at 4°C.
- Vortex. Draw up into a 20 or 30 ml syringe. Add a 25 guage needle and shear DNA by squirting through needle into fresh 50 ml conicals at 15 ml/conical.
- Add 0.9 ml/conical of 5 N NaCl.
- Boil in water bath (95 - 100°C) for 20 minutes.
- Cool in ice water.
- Vortex and then add 1 drop (from a standard transfer pipet) of 1.2 N HCl (concentrated HCl diluted 1/10) to neutralize.
- Add two volumes of 100% ethanol, let stand for 20 minutes on ice.
- Spin for 10 minutes in the Beckman GS-6R at 2500 rpm (4°C).
- Pour off the supernatant, let dry for 5 min
- Add 15 ml/tube of 0.02 M Tris, pH 7.6. Vortex.
- Allow to dissolve for several days at 4°C.
- Determine OD260 of a diluted aliquot to check concentration (1 OD260 = 50 µg DNA/ml), then store at -20°C.
This method is based, with permission, on an original protocol available here.