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Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176
785-532-5692 fax
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383

DNA DIGESTION AND SOUTHERN HYBRIDIZATION PROCEDURE FOR MONOCOT DNA

DNA Digestion

    1. Make a cocktail as follows:
Southern hybridization cocktail
10 X 1xbet best casino website
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 1xbet best casino website [minimum of 10 hours] in a 37°C oven.

Southern hybridization

  1. Stain 1xbet best casino website with 50 μL of 10 mg/mL ethidium bromide for 30 minutes and photograph.
  2. Treat 1xbet best casino website 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 1xbet best casino website 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 1xbet best casino website a glass plate over the top. 1xbet best casino website 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 1xbet best casino website on top of the wick sheet. Remove air bubbles as in Step 4.
  6. 1xbet best casino website gel on paper upper-surface down, then 1xbet best casino website 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. 1xbet best casino website a plastic strip between the two gel halves.
  8. Wet two '10 x 20 cm' pieces of chromatography paper and 1xbet best casino website on membrane. Remove air bubbles as in Step 4.
  9. Cover all areas, except that of the 1xbet best casino website , with plastic wrap to prevent evaporation and ensure that the capillary movement is only through the 1xbet best casino website .
  10. 1xbet best casino website a large stack of paper towels on top of the gel area. Put a glass plate over both stacks of paper towels and 1xbet best casino website a weight on top of the plate. Let the blot sit overnight [at least 20 hours].
  11. Rinse 1xbet best casino website with 2X SSC for 5–10 minutes. Use immediately or store wet.

Nylon transfer 1xbet best casino website : Hybond-XL nitrocellulose 1xbet best casino website , catalog # RPN203S; GE Healthcare, 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA; 1-800-526-3593;http://www5.gelifesciences.com.