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Star Republic: Guide for Biologists

Southern --- Chemiluminescence detection

This protocol uses the Genius Kit from Roche.

Labeling with digoxigenin-dUTP

1. 15 ml template DNA (0.5-1 mg) is denatured by boiling for 10 minutes and immediately placed on ice.

2. Add on ice, 2 ml hexanucleotide mix, 2 ml dNTP, and 1 ml Klenow enzyme.

3. Mix and centrifuge briefly.

4. Incubate at 37 C for 1 hour to overnight.

5. Add 1 ml 0.5 M EDTA.

6. Precipitate with ethanol (e.g., add 5 ml 7.5 M ammonium acetate and 60 ml cold ethanol) at -20 C for 1-2 hours.

7. Centrifuge at maximum speed for 10 minutes and wash the pellet with cold 70% ethanol.

8. Air dry and resuspend in 50 ml TE.

Gel electrophoresis and transfer to membrane

9. Run gel and transfer DNA to membrane as described in Southern --- radioactive probe.

Hybridization

10. Warm hybridization oven and solutions to 65 C.

11. Rinse the membrane in 2X SSC.

12. Pre-hybridization at 65 C for 1 hour to overnight in the pre-hybridization buffer (10% Genius Blocking Reagent in 0.1 M maleic acid and 0.15 M NaCl).

13. Boil the probe for 10 minutes.

14. Pour out the pre-hybridization solution.

15. Add hybridization solution (0.2% N-lauroylsarcosine, 0.1% SDS, 20% Genius Blocking Reagent in 5X SSC).

16. Add probe and hybridize for overnight.

The hybridization solution containing labeled probe can be re-used. To re-use, heat the hybridization solution to 95 C for 10 minutes.

17. Wash twice in 2X SSC/0.1% SDS for 10 minutes each.

18. Wash twice in 0.1X SSC/0.1% SDS for 10 minutes each at 65 C.

Detection

19. Rinse the blot in maleic acid buffer (0.1 M maleic acid and 0.15 M NaCl).

20. Wash the blot for 1 hour in the blocking buffer (10% Genius Blocking Reagent in 0.1 M maleic acid and 0.15 M NaCl).

21. Add AP-conjugate antibody (1:1,000) and rock at room temperature for 30 minutes.

22. Wash the blot twice with maleic acid buffer (0.1 M maleic acid and 0.15 M NaCl) for 15 minutes each.

23. Rinse the blot twice in the detection buffer (100 mM Tris-HCl pH9.5, 100 mM NaCl, 50 mM MgCl2).

24. Pour out the detection buffer and add 2 ml of CSDP (1:100 dilution in the detection buffer 100 mM Tris-HCl pH9.5, 100 mM NaCl, 50 mM MgCl2).

25. Incubate at 37 C for 15 minutes.

26. Drain off the CSDP solution and wrap the blot with Sarah wrap and expose to a X-ray film at room temperature.

Striping

27. Incubate in 0.5 mg/ml proteinase K, 0.1% SDS at 65 C for 1 hour.

28. Wash with 0.1% SDS, 5X SSC for 3x 5 minutes.

29. Incubate in 50% formamide, 10 mM sodium phosphate buffer (pH 6.5), at 65 C for 1 hour.

30. Rinse with 2X SSC.

Alternatively strip using NaOH.

27a. Incubate twice in 0.2 M NaOH/0.1% SDS at 37C for 15 minutes each.

28a. Rinse with 2X SSC.