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Star Republic: Guide for Biologists

Salmon sperm DNA

Virtualab Protocols (Chang Bioscience)
Salmon sperm DNA is frequently used as a blocking reagent in hybridizations. It needs to be sheared and denatured (by boiling) before use.

Sonication

1. Weight 0.5 g Salmon sperm DNA and dissolve in 50 ml TE (pH 8.0) overnight.

2. Sonicate until the solution is no longer sticky.

3. Centrifuge to remove debris.

4. Remove supernatant to a clean tube and precipitate DNA with two volumes of ethanol at -20 C or overnight.

5. Pellet DNA by centrifugation.

6. Wash the pellet with 70% ethanol, dry briefly and dissolve in 25 ml TE (pH 7.5).

7. Check concentration by OD measurement and dilute to desired concentration.

8. Aliquot and store at -20 C.

NaOH and boiling

1. Weight 0.5 g Salmon sperm DNA and dissolve in 50 ml 0.4 M NaOH overnight.

2. Place the sample in a boiling water bath, boil for at least 45 minutes.

3. Cool on ice.

4. Neutralize with glacial acetic acid to pH 7.0.

5. Centrifuge to remove debris.

6. Remove supernatant to a clean tube and precipitate DNA with two volumes of ethanol at -20 C or overnight.

7. Pellet DNA by centrifugation.

8. Wash the pellet with 70% ethanol, dry briefly and dissolve in 25 ml TE (pH 7.5).

9. Check concentration by OD measurement and dilute to desired concentration.

10. Aliquot and store at -20 C.

Shearing through fine needle

1. Weight 0.5 g Salmon sperm DNA and dissolve in 100 ml TE (pH 7.5) overnight.

2. Draw up the solution into a syringe, add a 25 gauge needle. Shear DNA by squeezing through needle into a fresh tube.

3. Add 1.8 ml 5 M NaCl.

4. Boil the DNA sample in a boiling water bath for 45 minutes.

5. Cool on ice.

6. Vortex and then neutralize to pH 4-7 by adding 1 drop of diluted HCl (1:10 from the concentrated hydrochloric acid).

7. Centrifuge to remove debris.

8. Remove supernatant to a clean tube and precipitate DNA with two volumes of ethanol at -20 C or overnight.

9. Pellet DNA by centrifugation.

10. Wash the pellet with 70% ethanol, dry briefly and dissolve in 25 ml TE (pH 7.5).

11. Check concentration by OD measurement and dilute to desired concentration.

12. Aliquot and store at -20 C.