DIG Southern Blotting

 

Smith and Summers, 1980. Anal. Biochem . 109: 123-129.

1. Digest 10-15 µg genomic DNA with desired restriction enzyme overnight at 37^o C.
2. Run digest on a 1% TBE agarose gel at 100V until 1^st blue dye reaches the bottom of gel.
3. Stain gel with EtBr and take a photograph (use ruler in photo for later size determination).
4. Gently rock the gel in 2 volumes (~250mls) of 0.25M HCl for 10-15 minutes (until loading buffer dyes change color ― 1^st blue turns yellow).
5. Rinse the gel in distilled water for 5 min.
6. Denature the DNA by gently rocking the gel in 2 volumes of 1.5M NaCl, 0.5M NaOH for 10-15 min.
7. Repeat step 6.
8. Rinse the gel in distilled water for 5 min.
9. Neutralize the gel by gently rocking in 1M ammonium acetate, 0.02M NaOH for 15-30 min.
10. Repeat step 9.
11. Rinse gel in 20X SSC for 10 min., do not discard solution.
12. Flip the gel (DNA side up) on a piece of seran wrap on the bench. Wet the nylon membrane (Magnacharge) and Whatman paper in the 20X SSC that remains.
13. Set up transfer without a SSC resevoir, let stand overnight.

Hybridization

Prehyb: Incubate blot with 30mls of DIG Easy Hyb (Roche) for at least 1 hr at 42^o C. Prehyb solution can be reused---save in a 50ml conical tube.

Hybridization

With a New Probe

1. Boil probe for 10 min. and chill on ice.
2. Use ~500 ng of labeled probe DNA/blot (1µ l/ml)
3. Add probe to 30mls of Easy Hyb solution prewarmed to 42^o C.
4. Remove prehyb from blot.
5. Add diluted probe to blot.
6. Hybridize overnight at 42^o C with gentle agitation.

With a Recycled Probe

1. Thaw
2. Heat at 68^o C for 10 min. to denature.
3. Move to step four above.

Wash

1. Remove probe from blot, save in a 50ml conical tube (freeze)
2. Wash for 5 min. in 2X SSC, 0.1% SDS at room temperature.
3. Repeat step 2.
4. Wash for 15 min. in prewarmed 0.5X SSC, 0.1% SDS at 68^o C.
5. Repeat step 4.

Detection

1. Rinse membrane in maleic acid buffer for 5 min. with shaking at RT.
2. Dilute 10X Blocking buffer in maleic acid buffer to 1X.
3. Block membrane for 1-3 hours with shaking at RT.
4. Cetrifuge anti-DIG Ab for 1 min.
5. Dilute Ab 1:10,000 in fresh blocking buffer.
6. Incubate for 30 min. at RT with gentle shaking.
7. Wash for 15 min. in Wash Buffer 3.
8. Repeat step 7.
9. Rinse in detection buffer for 5 min.
10. Cut a piece of Seal-A-Meal bag larger then the blot, open one side.
11. Lay the membrane on one side of bag, only touching the corners of the blot, or by using forceps. On the other place several drops of CSPD solution (covering the area of the blot).
12. Lift the membrane side onto the CSPD side and push out any air bubbles.
13. Incubate at RT for 5 min. No shaking.
14. Press out excess CSPD and seal the bag using the Seal-A-Mealer.
15. Incubate at 37^o C for 30 min. to enhance signal.
16. Expose film--initially for 10 min.