Day 1:

• Determine the approximate concentration of your DNA sample by spectrophotometry or by running a portion of your sample on a gel (usually ~5 micro litre).
• Label a 1.5 ml microfuge tube for each digestion.
• Prepare a digestion mixture for each sample in the labeled 1.5 ml tube:

  	Volume
  H2O	(15.5 -x) micro litre
  10X React 3 buffer	2 micro litre
  spermidine (10 µg/micro litre)	1 micro litre
  15 to 20 µg genomic DNA	x
  Enzyme (3-5 unit/µg of DNA)	micro litre
  Rnase A (10 mg/ml)	0.5 micro litre

  1(spermidine should be prepared at least once a year and frozen at -20 C)

  2(amount of DNA may have to be calibrated, the DNA extraction method we use probably includes RNA contaminants, thus resulting in an overestimated DNA concentration. Lower amounts of DNA extracted through kits can be used)

  Total volume = 20 micro litre

• Mix and spin down briefly. Place the 1.5 ml tubes in the 37 C incubator for at least 4 hours.
• Add running dye to each sample (2 micro litre), load onto 0.8% agarose gel (use 170 ml agarose for our gel mold (15X20 cm)) and run overnight at 30-40 V.

   

  Day 2:

• After running gel, take photograph, then depurinate DNA by soaking gel in 0.25M HCl (~300 to 500 ml) for 10 min. with shaking at room temperature in a glass baking dish. Rinse gel briefly with dH2O.
• Denature DNA by soaking gel in 300-500 ml of 1X Southern base solution at room temperature with shaking for 15 minutes. Pour off solution and soak gel in another volume (300-500 ml) of 1X Southern base solution for 15 minutes.
• Pour about 500 ml of 1X Southern base solution into a glass baking dish. Place a piece of glass or plastic across the dish to use as a support.
• Place a piece of gel blot paper (pre-wetted with 1X Southern base solution) across the glass support, and allow it to hang into the base solution. This will act as a wick.
• Place the gel on top of the wick, and roll it with a pipette to remove air bubbles.
• Cut a piece of nylon membrane (about the same size as the gel) and soak it in 1X Southern base solution. We use Hybond N+ membrane. Place the membrane on top of the gel.
• Cover the edge of the membrane to the edge of the dish with saran wrap on all four sides of the gel.
• Place three pieces of gel blot paper on top of the membrane (wet the first paper in Southern base solution). Then put a stack of paper towels on top of the gel blot paper.
• Finally, place a plastic dish with ~500 ml of H2O on top of the entire thing.
• Let the DNA transfer 3-6 hours. Then, remove the membrane from the gel and place in ~200 ml 1M Tris pH 7.5, 1.5M NaCl and shake for 2-3 minutes to neutralize the base solution.

• Place membrane on blot paper to remove excess buffer, then crosslink DNA using UV crosslinker (Stratagene). Blot can be stored between two sheets of blotting paper in a plastic bag at –20 C or –80 C until ready for hybridization.

Pre-hybridization and hybridization of blot (this protocol assumes ~300 cm² size blot)

• Preheat ~50 ml of hybridization buffer to ~60 C in the hybridization oven.
• Pour hybridization buffer into a plastic container (slightly larger than the blot). Submerge blot in the hybridization buffer, blot should be completely wet.
• Roll up blot (DNA side "inside") and place in a hybridization tube. Pour hybridization solution from the container to the hybridization tube. Secure cap. Unroll blot inside the tube by rotating the tube.
• Place tube with blot into the hybridization oven and incubate at 60 C for 30 min.
• Prepare salmon sperm DNA: place 250 micro litre in a screw cap microfuge tube, incubate in a boiling water bath or heat block for 5 min. then on ice for 5 min.
• After the 30 min. 60 C incubation period for the blot (see step 21), add the 250 micro litre denatured salmon sperm DNA to the hybridization buffer in the tube. Incubate the blot for an additional hour at 60 C.

Prepare probe for labeling:

• Prepare a 1/5 dilution of the cross-linker reagent (supplied with the kit): add 10 micro litre of cross-linker reagent to 40 micro litre water. Place on ice until ready for use.
• Our probe DNA is prepared by PCR , followed by gel extraction with a Qiagen kit. A typical DNA concentration of ~50 ng/micro litre is obtained; therefore, 5 micro litre of gel-purified PCR product is added to 20 micro litre water to obtain the proper amount and concentration of DNA.)
•  Heat the probe DNA (25 micro litre for our typical case) in boiling water 5 min. or in a heat block at 100 C, then on ice for 5 min., then spin down briefly.
• Add 25 micro litre of reaction buffer (supplied with kit) to the denatured probe DNA, mix gently, then back on ice (total volume = 50 micro litre).
•  Add 5 micro litre of labeling reagent (supplied with kit), mix gently, then back on ice.
• Add 25 micro litre for our typical case of working dilution (1/5) of cross-linker reagent, mix gently, spin down contents briefly.
• Incubate reaction mix for 30 min. in 37 C water bath or heat block.
• Place probe on ice until ready for use (up to 2 hours is fine).
• After prehybridization is complete (90 min. prehyb. total), add PCR product labelled probe (~80 micro litre total) directly to the hybridization buffer (best to remove hyb. solution to 50 ml tube, add probe, mix briefly, then add back to the hybridization tube).
• Hybridize overnight in hybridization oven at 60 C.

Post hybridization washes and blot development

• Preheat primary wash buffer (~1 liter) in a water bath or microwave oven to 60 C. Prepare secondary wash buffer by adding 50 ml of 20X stock secondary buffer to 950 ml water and 2 ml of 1M MgCl₂ solution. The secondary wash buffer is used at room temperature.
• Transfer blot from hybridization tube into a clean plastic container with lid (DNA side up), wash two times in primary wash buffer (500 ml, 60 C, 10 min. each wash).
• Transfer blot to clean plastic dish (DNA side up), wash two times in secondary wash buffer (500 ml, room temperature, 5 minutes each wash).
• Remove blot from the secondary wash buffer, and "wick off" excess buffer by touching a corner of the blot to the side of the plastic dish (~10 sec.), then transfer blot DNA side up to a clean, dry plastic dish (size similar to blot). Pipette ~8 ml (~0.025 ml/cm²) of CDP-star reagent (provided with the kit) onto the blot, try to cover the blot as evenly as possible. Tilt the dish with blot back and forth to distribute the CDP-star to all areas of the blot, then incubate ~4 min. at room temperature.
• Remove blot from CDP-star, "wick off" excess CDP-star by touching the corner of the blot to the side of the plastic dish (~10 sec.). Then place the blot inside a clean "sheet saver" plastic sleeve (some trimming might be required) and then into a film cassette. The blot will still be "shiney" wet with CDP-star when placing in the plastic sleeve---this is normal. Drying or blotting on filter paper should not be performed.
• Incubate the blot in the cassette for ~1 hr. at room temperature, then place a film on for 10-15 min., then develop. Film exposure times may have to be varied to obtain the best results (we used X-omat blue film for our test cases).