Kansas State University

DNA DIGESTION AND SOUTHERN HYBRIDIZATION PROCEDURE FOR MONOCOT DNA

DNA Digestion

1. 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

1. Add 15 μL [20–25 μg] DNA to each tube containing 20 μl of the cocktail. Pipette several times to mix.
2. Incubate the samples overnight [minimum of 10 hours] in a 37°C oven.

Southern hybridization 

1. Stain gel with 50 μL of 10 mg/mL ethidium bromide for 30 minutes and photograph.
2. 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].
3. 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].
4. 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.
5. 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.
6. 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.
7. Place a plastic strip between the two gel halves.
8. Wet two '10 x 20 cm' pieces of chromatography paper and place on membrane. Remove air bubbles as in Step 4.
9. 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.
10. 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].
11. 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.