Southern Blotting of Genomic DNA-

1. Digest DNA:
• 1.5 μg DNA in a 25 μI reaction containing BSA
• 3 to 5 units for 5 to 3 hours (aim for 10-fold overdigestion; remember many enzymes will not survive well for more than 3 hr).
• Also prepare molecular weight standard (Lambda plus HindIII); avoid plasmid-derived markers (1 kb ladder, etc.) since it might cross-hybridize with the probe!
2. Run gel:
• Ioad samples onto agarose gel (typically 0.8 to 1.0% agarose in TBE, depending on desired size range).
• run gel at maximum rate of 5 volts per cm.
• run until the bromophenol blue has run ca. 9 cm (some DNA will run 1-2 cm below the dye).
• Using gloves, place in staining solution (0.5 to 1 μg/ml ethidium bromide in water, 20-40 min. Be careful not to break the gel.

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