Alkaline Southern Blotting Procedure

Author: Suzanne Gerttula
Date: March 14,1994

1. Digest between 1 and 10 ug Genomic DNA. I digest at 37 C for about six hours over the course of a day. Spin down briefly, heat to 65 C for 10 min and then chill on ice. When ready to load, add an appropriate amount of 10X tracking dye, no EtBr.
2. Pour the gel: Mix 2 grams ultra pure agarose in 250 ml 1X TAE (.8%). Heat in microwave to boiling. Continue boiling and swirling until agarose is completely dissolved. Cool gel solution on ice stirring constantly to aprx 50 C. Pour into wide (20 cm) gel rig.
3. Prepare DNA size markers for loading in gel:

   DNA H2O SALT TRACKING DYE 1 Kb ladder, 250 ug/245 ul 5.0 ul 4.0 ul 1 ul 1 ul 100 bp ladder, 200 ug/ml 5.0 ul 4.0 ul 1 ul 1 ul

4. Submerge gel in 2000 ml 1X TAE
5. Load samples into wells. Electrophorese at 40 Volts, 35 mAmps for 16 hours.(Overnight)
6. Post stain gel in .5 ug/ml EtBr for 20 min.
7. Photograph with fluorescent ruler 1/8th sec with ruler and an additional 1 sec on gel alone.
8. Invert gel, place into .25M HCl for 7 min.
9. Rinse briefly in dH2O.
10. Soak in 0.3M NaOH,0.3M NaCl for 10 min.
11. Set up a transfer tray (9 X 12 inch glass baking dish) by pouring transfer solution in the tray (about 1000 ml) and placing a glass plate that is wide enough to span the width of the tray over top.
12. Cut Zeta Probe GT membrane to size of gel excluding wells. Cut a "wick" out of 3mm paper. This should be the same width as your gel and long enough to drape over the glass plate and well into the transfer solution. Cut three pieces of 3mm paper the size of the gel excluding wells.
13. Prewet 3mm wick in transfer solution. Drape it over the glass plate allowing ends to dangle in the solution. Squeeze out air bubbles by rolling a clean 10ml pipet over the surface.
14. Place inverted gel onto the 3mm wick. Squeeze out air bubbles.
15. Prewet membrane in H2O then in transfer solution. Place onto gel with the top just below the wells, squeeze out air bubbles.
16. Prewet the two 3mm sheets cut to size of gel in transfer solution. Place onto membrane. Squeeze out air bubbles. Seal around the edges of the blot by laying down strips of plastic.
17. Place blotting material on top, weight it down with a glass plate and a tray of water or other material weighing about .5 Kg. Prevent evaporation of the transfer solution by sealing with parafilm on either end of the glass tray. Let transfer overnight.
18. Next day, take off blotting material and mark the position of the wells.
19. Remove the membrane and rinse in 200ml: 0.1M Tris Buffer pH 7 15 min. 1.0M NaCl
20. Blot dry on 3mm paper. Bake in 80 C vacuum oven for 90 min.

    PREHYBE SOLUTION: 2% SDS 0.25 M Na Phosphate buffer pH 7.2 0.5 mg/ml Herring Sperm DNA HYBE SOLUTION: 2% SDS 0.25 M Na Phosphate buffer pH 7.2 0.5 mg/ml Herring Sperm DNA 5% Dextran Sulfate

21. Prehybe the blot in about 20 ml of prehybe solution for 1/2 hour at 65 C. Try to keep everything at temp. by working quickly to add or change solns.
22. Hybridize the membrane using a probe with counts between 1 %26amp; 5 X 10 7cpm total. After spinning the random prime labelling reaction over a sephadex G-50 column, make the solution 1% with respect to SDS then place at 95 C to denature, 5 Min. Do not quench on ice, add the 95 C probe directly to 10 ml of 65 C hybe solution. Discard the prehybe solution and add the hybe solution. Hybridize at 65 C overnight.

    WASH STEP: High Stringency 20mM Sodium Phosphate buffer pH 7.2 Two changes at 65 C, 50 ml, 1% SDS 30 min ea. Two changes at 65 C, 200 ml, 30 min ea. Medium Stringency (Equivalent to .6X SSC) 100mM Sodium Phosphate buffer pH 7.2 Two changes at 65 C, 50 ml, 1%SDS 30 min ea. Two changes at 65 C, 200 ml, 30 min ea. Low Stringency 250mM Sodium Phosphate buffer pH7.2 Two changes at 65 C, 50 ml, %SDS 30 min ea. Two changes at 65 C, 200 ml, 30 min ea. When the final wash step is complete, do not blot dry your membrane. Place it wet into a seal-a-meal bag and seal. Expose to X-Ray film.

    SOLUTIONS: 10X TRACKING DYE: 0.25% Bromophenol Blue 0.25% Xylenecyanol FF 15% Ficoll (type 400, Pharmacia) in water 50X TAE: 2M Tris-acetate, 50mM EDTA. To 600 ml dH2O add 242 g Tris base, 100 ml 0.5M EDTA pH 8.0, and 57.1 ml glacial acetic acid. Bring up to 1 liter with dH2O. 1M PHOSPHATE BUFFER: MAKE 1M DIBASIC SODIUM PHOSPHATE: Dissolve 187.6 grams Na2HPO4 Heptahydrate into 700 ml TV Heat and stir until dissolved. MAKE 1M MONOBASIC SODIUM PHOSPHATE: Dissolve 34.5 grams NaH2PO4 monohydrate into 250 ml TV Stir until dissolved. Mix 700 ml dibasic with 230 ml monobasic Bring the solution to pH 7.2 by adding 10M NaOH. 0.3M NaOH,0.3M NaCl: Dissolve 12 grams NaOH + 17.5 grams NaCl into 1000 ml TV 0.1M Tris pH7,1.0M NaCl: Mix 20 ml 1M Tris pH7 + 40 ml 5M NaCl in 200 ml TV HYBE/PREHYBE: 12 ml 10% SDS 15 ml 1M Phosphate Buffer pH 7.2 3 ml 10 mg/ml Herring Sperm DNA 30 ml H2O 60 ml Total Volume Mix the SDS, the Phosphate Buffer and the H2O and heat to 65 C. Meanwhile, boil the Herring Sperm DNA for 10 min. Add the Herring Sperm hot to the 65 C mix. Aliquot 10 ml of this for each hybe solution and add 0.5 grams dextran sulfate. Keep at 65 C and mix occationally until the dextran sulfate goes into solution. ZETA-PROBE MEMBRANE: Obtained from Bio-Rad Laboratories 3300 Regatta Boulevard Richmond, CA 94804 800-424-6723