Preparation of Sonicated Salmon Sperm DNA

Procedure:

 

 

 

1) 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.

 

 

 

2)   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.  To this add 0.9 ml/conical of 5 N NaCl.

 

 

 

3) Boil in water bath (95 - 100°C) for 20 min.  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.

 

 

 

4)   Add two volumes of 100% ethanol, let stand for 20 min on ice, then spin for 10 min in the Beckman GS-6R at 2500 rpm (4°C).  Pour off the supernatant, let dry for 5 min, then add 15 ml/tube of 0.02 M Tris, pH 7.6.  Vortex and let 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.