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Star Republic: Guide for Biologists

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1. Digest appropriate amount of DNA (1-10 mg for genomic DNA and less for clones) and run agarose gel.

2. Take a gel picture with a ruler along the side and mark the gel orientation (e.g., by cutting on corner).

3. (Optional) Depurination: soak the agarose gel in 0.25 M HCl for 10 minutes. May be required for transfering DNA fragments longer than 5 kb.

4. Denature: soak the agarose gel in 0.5 M NaOH for 10 minutes.

5. (Optional) Neutralization: soak the agarose gel in 0.25 M Tris-HCl (pH 7.4) for 10 minutes.

6. Transfer the DNA to a membrane using capillary transfer or vacuum blot.

7. After transfer, gently peel the membrane from the surface of the gel, rinse it in 2x SSC, and air dry.

8. Cross-link DNA to the membrane by illuminating under a UV source or use a cross-linker. For nitrocellulose membranes, bake for 2 hours at 80 C in a vacuum oven.

9. Pre-hybridize in 10-20 ml of hybridization buffer (5X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml Salmon sperm DNA).

10. Hybridize with 32P or 33P labeled probe in 10 ml of hybridization buffer (5X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml Salmon sperm DNA).

11. Hybridize at 68 C for overnight.

12. Wash in 200-500 ml washing buffer I (2X SSC, 0.1% SDS) twice for 5-10 minutes each with gentle shaking.

13. Wash in 200-500 ml pre-warmed washing buffer II (0.2X SSC, 0.1-0.5% SDS) for 10-30 minutes at 68 C for 10-30 minutes.

14. Repeat step 13 for 1-2 times.

15. Wrap the membrane in a Saran wrap.

16. Expose to a X-ray film. If using an intensifying screen, place the cassette in -70 C.

17. Exposure time varies from 30 minutes for plasmid DNA to overnight or several days for genomic DNA.