Preparation of Slot Blot Filter

1. Add 200 pL 0.3M NaOH to 5 pg of plasmids containing genomic or cDNA clones of interest (see Note 5).

2. Boil for 10 min, and quench on ice.

3. Add 400 pL 5M NH4 acetate. Apply to a nylon hybridization membrane (e.g., Amersham Hybond-N), previously equilibrated with 2X SSPE, using a slot-blot applicator.

4. Rinse the filter in 2X SSPE. Air-dry and then bake at 80°C for 60 min.

5. Covalently crosslink the DNA to the matrix by exposure, DNA side down on Saran™ wrap, to a UV light (312 nm) transilluminator for 2 min (see Note 6).

6. Rinse the filter in wash buffer A.

7. Prehybridize in hybridization buffer at 65°C for at least 5 min.

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