Southern Hybridization

3.7.1. Prewarm the Prehybridization Buffer to 65°C

1. Use 5 mL in a small (150 o 35-mm) hybridization bottle.

2. Place the blot in a prewarmed hybridization bottle and rinse with 2X SSC (pre-warmed to 65°C), making sure that the DNA side of the membrane faces in and that all air bubbles between the blot and the bottle are removed.

3. Discard the 2X SSC from the bottle.

4. Add the prehybridization buffer (prewarmed to 65°C) to the bottle.

5. Add denatured sheared salmon sperm DNA to the prehybridization buffer for a final concentration of 100 ^g/mL.

6. Prehybridize at 65°C with medium speed rotation in a hybridization oven for at least 1 h.

3.7.2. Radiolabeling of the Gel-Purified Probe is Adapted from the Guide to Molecular Cloning Techniques (16)

1. Denature a 10-ng probe template diluted in dH2O to 10 ^L vol by first sealing in a glass capillary tube.

2. To do this, touch the tip of the tube to the template solution: the solution should be taken up into the tube by capillary action.

3. Next, center the solution in the tube by tipping the tube to one end.

4. Seal the ends of the tube by holding each end, sequentially, in the hottest part of a Bunsen burner flame for about 3 s.

5. Let the sealed ends cool; then test the seals by applying negative pressure to each with a pipet bulb.

6. Finally, boil the sealed tube of probe template for 5 min submerged in a small beaker, and then chill in an ice water bath for 5 min.

7. Dry off the capillary tube and carefully score it about 2 cm away from the centrally contained liquid on both sides using a diamond tip scriber; then snap the capillary at each score line.

8. Use the supplied pipet bulb to apply a small amount of pressure to eject the denatured probe template solution into a chilled microfuge tube containing: 1 ^L of 10 mg/mL BSA, 11.5 ^L of LS solution (440 mM HEPES-NaOH, pH 7.6, 44 ^M each of dTTP, dCTP, and dGTP, 110 mM Tris-HCl, pH 7.5, 11 mM MgCl2, 22 mM 2-mercaptoethanol, and 300 ^g/mL random deoxyribonucleotide hexamers).

9. Add 50 ^Ci [a-32P]dATP (3000 Ci/mmol) and 2.5 U Klenow fragment for a total volume of 30 ^L.

12. Add 1 ^L 0.5 M EDTA, pH 8.0, to stop the reaction.

13. This will yield approximately 35 ng of radiolabeled probe.

3.7.3. Testing the Specific Activity of the Labeled Probe

1. Make a 1/16 dilution of the reaction mixture in 0.1 N NaOH.

2. Spot 1 ^L three times on a piece of dry blotting membrane.

3. Let spots dry, and then UV crosslink the membrane.

4. Wash the membrane three times in 2X SSC in a hybridization bottle at 65°C to remove unincorporated [a-32P]dATP.

5. Each spot should be no lower than 200 counts/s to get a useable signal on the hybridized blot.

6. Make a record of the test spots by exposing to Kodak Biomax MR X-ray film for 15 min at room temperature.

7. Figure 3 shows the results obtained from several probe labeling reactions.

3.7.4. Hybridization

1. Add 5 ^L of 10 mg/mL sheared salmon sperm DNA to the labeled probe reaction and mix well in a microfuge tube.

2. Add dH2O to a 250 ^L vol. Make sure to poke a small hole into the lid of the tube to prevent the lid from bursting open upon heating.

3. Boil the reaction for 10 min in a beaker using a hot plate, taking care to not dilute the contents of the tube.

4. Chill on wet ice for 15 min.

5. Spin down briefly in a microfuge tube.

6. Add the denatured labeled probe reaction mix to the prehybridization buffer with the prehybridized blot for a final concentration of 5 ng/mL.

7. Higher probe concentrations may lead to nonspecific hybridization.

8. Hybridize from 4 h to overnight at 65°C.

9. After hybridization, discard the hybridization buffer from the bottle (into the liquid radioactive waste).

10. Wash the blot once for 15 min at 65°C with maximum speed rotation in pre-warmed 2X SSC (use at least 10 times the hybridization buffer volume for all washes).

11. Wash twice under the same conditions in prewarmed 2X SSC containing 1% SDS, once in prewarmed 0.1X SSC/0.1% SDS at the same speed, 63°C for 30 min (the

Detecting Nucleosome Ladders Sample: A

Spot no. 1

Spot no. 2

Spot no. 3

Counts/sec: 50



Fig. 3. Testing the specific activity of the labeled probe. Small portions of each probe-labeling reaction mixture (A, B, C, or D) were diluted 16-fold with 0.1 N NaOH, and 1 ^L of each diluted mixture was spotted on a piece of nylon hybridization membrane in triplicate (Spot nos. 1, 2, and 3). The spots were dried and UV-crosslinked, and the membrane was washed three times with 2X SSC in a hybridization bottle at 65°C to remove unincorporated [a-32P]dATP. The specific activities of the probes were estimated using a Geiger counter; the 1000 Counts/s spots correspond to approx 109 counts/min per ^g of DNA. The membrane was then exposed to X-ray film for 15 min.

high-stringency wash) and once with vigorous shaking on a shaker at room temperature in 0.1X SSC for 15 to 30 min (see Note 5).

12. The final wash is to remove radioactive particles trapped on the blot that may give black spots on the autoradiogram.

13. Monitor the activity of the washes after each wash. The activity should decrease to almost background levels by the third wash. If activity increases significantly in the fourth wash, this is usually a sign of a nonspecifically hybridized probe.

14. Also monitor the activity of the blot background and edges, where no probe should be hybridizing. Additional washes may be necessary to further reduce background.

15. Wrap the washed blot wet in plastic wrap. Try to avoid creases and bubbles in the plastic that can affect the exposure.

16. Radioactivity levels of 5 to 10 counts/s usually need an overnight (16-h) exposure at -70°C using a Kodak Biomax MS intensifying screen.

17. Keeping the blot wet allows for additional washes of the blot if necessary or stripping of the probe from the blot and subsequent rehybridization of the blot. Drying the blot will irreversibly fix the probe to the membrane.

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