DNA DIGESTION AND SOUTHERN HYBRIDIZATION PROCEDURE FOR MONOCOT DNA
- Make a cocktail as follows:
Southern hybridization cocktail 10 X buffer 3.50 µL BSA [stock: 10 mg/mL] 0.35 µL ddH20 5.65 µL RNase [stock: 100 mg/mL] 0.50 mL EcoRI [12 units/µL] 10.00 µL TOTAL 20.00 µL/tube
- Add 15 μL [20–25 μg] DNA to each tube containing 20 μl of the cocktail. Pipette several times to mix.
- Incubate the samples overnight [minimum of 10 hours] in a 37°C oven.
- Stain gel with 50 μL of 10 mg/mL ethidium bromide for 30 minutes and photograph.
- Treat gel with 1 L of 0.25M HCl for 25 min at room temperature with gentle shaking [the color of the loading buffer will become a bright yellow].
- Rinse gel twice with distilled water and treat with 1 L 0.4M NaOH for 20 min with gentle shaking [The color of the loading buffer will return to blue].
- Fill a glass dish with 2 L 0.4M NaOH and place a glass plate over the top. Place a '23 x 30 cM' piece of 3MM chromatography paper over the plate so that the ends are immersed in the liquid [the ‘wick’]. Wet the paper and remove all air bubbles by rolling a glass pipette over the paper.
- Wet two '22.5 x 22.5 cm' sheets of chromatography paper with 0.4M NaOH and place on top of the wick sheet. Remove air bubbles as in Step 4.
- Place gel on paper upper-surface down, then place a sheet of nylon membrane on the gels. Trim gel to the exact size of the membrane. Remove any air bubbles as in Step 4.
- Place a plastic strip between the two gel halves.
- Wet two '10 x 20 cm' pieces of chromatography paper and place on membrane. Remove air bubbles as in Step 4.
- Cover all areas, except that of the gel, with plastic wrap to prevent evaporation and ensure that the capillary movement is only through the gel.
- Place a large stack of paper towels on top of the gel area. Put a glass plate over both stacks of paper towels and place a weight on top of the plate. Let the blot sit overnight [at least 20 hours].
- Rinse membrane with 2X SSC for 5–10 minutes. Use immediately or store wet.
Nylon transfer membrane: Hybond-XL nitrocellulose membrane, catalog # RPN203S; GE Healthcare, 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA; 1-800-526-3593; http://www5.gelifesciences.com.