Preparation of Salmon Sperm DNA

Denatured Salmon Sperm DNA is used in prehybridization and hybridization solutions to reduce background signal.The Salmon Sperm DNA (Sigma No.D-1626)is first "denatured" in large volumes and then again immediately before it is used.Two methods (Sonication or NaOH/boiling SS DNA)are prevail.NaOH/boiling SS DNA is available in this lab.

NaOH/boiling of SS DNA

1.Add 1.0 g salmon sperm DNA to 100 ml 0.4 NaOH in a 250 ml Erlenmyer flask: (If 250-ml centrifuge bottles are necessary,ask Dr.M.D.Thomas to use his centrifuge and 250 ml centrifuge bottles).Dissolve by placing on a rotator or stir plate overnight at room temperature.

2.Place in a boiling water bath for 45 min to shear the DNA.Chill on ice.

3.Neutralize with glacial acetic acid to pH 7.0.

4.After transfer 50 ml PPT,and centrifuge tubes (2000 rpm,10 min)to remove debris.Pour DNA solution into another 250 ml Erlenmyer flask.

5.Add two volume of 95 % EtOH,and place at -20℃ for 1 hr.

6.Transfer to PPT,and centrifuge tubes again (2000 rpm,10 min).

7.Rinse pellet with 70 % EtOH,invert to the tube to drain for 1-2 min,if desired,vacuum dry 5 min,dissolve in 50 ml TE.

8.Determine DNA quantity by determining the fluorescent density or measuring the absorbance of the solution at 260 nm and dilute to 10 mg/ml.

9.Store at -20℃.

NaOH denaturation of SS DNA immediately before use

1.Remove the appropriate amount of previously NaOH/boiled SS DNA after thawing.

2.Add 1/10 volume of 1 N NaOH,voltex and let sit for 10 min for room temperature.

3.Neutralize the base with 1/10 volume of 1.8 M Tris-HCl and 0.2 M Tris^- Base sequentially.Vortex and let sit at room temperature for 10 min.

4.Add prehybridization or hybridization solution and mix in a stir plate.