| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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| Southern blotting provides a means for determining the existence of particular sequences or genes within genomic DNA. Genomic DNA is digested and then separated by agarose gel electrophoresis. The DNA is then transferred to a nylon or nitrocellulose filter. After fixing the DNA to the filter, the filter is probed with radiolabeled DNA complementary to the DNA sequence of interest. Autogradiography indicates DNA bands the probe hybridized to. |
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1. Run an agarose gel with the desired samples of digested genomic DNA and DNA markers and stain it with Ethidium Bromide (CAUTION! see Hint #1; see protocol on Agarose Gel Electrophoresis of DNA).
2. Visualize the DNA using a long-wavelength UV light box and cut the gel to size (see Hint #2). Photograph the gel next to a ruler for reference.
3. If DNA fragment target is larger than 15 Kb, incubate the gel with shaking in 500 ml of 50 mM HCl for 20 min in a Pyrex dish.
4. Denature the DNA by incubating the gel in 500 ml Denaturing Solution for 30 min with shaking.
5. Neutralize the gel in 500 ml of Neutralization Buffer for 45 min with shaking and with one change of buffer at 30 min.
6. Cut 3 pieces of Whatman 3MM the size of the sponge used for the transfer (usually 23 x 13 cm). Cut 3 pieces of Whatman 3MM to the size of the gel.
7. Wearing gloves, cut 1 piece of Hybond-N™ membrane (Amersham) the size of the gel (or 1 to 2 mm larger). Cut paper towels the size of the gel to make a 10 cm stack.
8, Prewet the membrane by floating it on ddH2O. When the underside surface is completely wetted, submerge the membrane in 10X SSC for 5 min.
9. Fill a long Pyrex dish with 1 liter of 20X SSC. Place the sponge in the solution and wet the 3 pieces of Whatman 3MM paper the size of the sponge with 20X SSC.
10. Lay the wetted Whatman 3MM papers on the sponge and press out any air bubbles with a glass rod or glass pipette.
11. Notch a corner of the gel and carefully place the gel upside down on top of the Whatman 3MM paper. Press out any air bubbles with a glass rod or glass pipette.
12. Remove excess liquid from the top of the gel with a gloved finger and then lay the prewetted Hybond-N™ membrane very carefully on the gel. Press out any air bubbles with a glass rod or glass pipette (see Hint #3).
13. Mark the filter with an ink pen for orientation. Presoak the 3 pieces of Whatman 3MM the size of the gel in 10X SSC and layer them on top of the membrane. Press out any air bubbles with a glass rod or glass pipette.
14. Stack the paper towels on top of the Whatman 3MM and cover the exposed surface of the sponge and the Pyrex dish with plastic wrap to reduce evaporation of the 20X SSC (if desired). Make sure that none of the paper towels in the stack come in contact with the sponge or filter paper below the gel; this will short circuit the transfer.
15. Top the transfer stack with a glass plate and a 500 g weight.
16. Allow the transfer to occur overnight.
17. Disassemble the transfer stack and peel the filter away from the gel with blunt forceps.
18. Crosslink the DNA to the Hybond-N™ membrane in a Stratalinker (Stratagene) set to "Autocrosslink" (120,000 μJ or 120 mJ).
19. Rinse the membrane for 30 sec in 2X SSC to remove the agarose and wick away excess liquid (see Hint #4).
20. Roll up the filter with the DNA side facing on the inside. Put the filter into hybridization oven bottle with 2X SSC, pour off the buffer, and roll bottle on a table to unroll the filter so it coats the side of the bottle without bubbles (see Hint #5).
21. Add 15 to 30 ml of Hybridization Buffer to the bottle and prehybridize the filter for 1 hr at 65°C in the rotisserie hybridization oven (see Hint #6).
22. Add 1 million cpm/ml of denatured probe to the bottle and hybridize the membrane overnight (see Protocols for Labeling Probe DNA and Hint #7).
23. Pour off hybridization buffer and wash the membrane twice for 20 min each in preheated (65°C) Wash Buffer, filling the bottle half full with buffer and rolling at 65°C in the hybridization oven.
24. Remove the membrane from the bottle and incubate the membrane in 500 ml of Wash Buffer in a plastic box at 65°C with shaking for 20 min.
25. Allow the filter to air dry briefly. Do not completely dry the filter if it is to be stripped and reprobed. Enclose the membrane in plastic wrap and expose the blot to film using an intensifying screen.
26. To strip the blot, incubate the membrane with shaking in 0.4 M NaOH at 42°C for 30 min. Then incubate the membrane in Stripping Solution at 42°C for 15 min.
Then proceed with prehybridization as above (Step #21) and hybridize with another probe.
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| Wash Buffer |
| 0.1% (w/v) SDS
0.1X SSC
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| 0.4 M NaOH |
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| 1 M Tris-HCl, pH 8.0 |
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| 1 M NaOH |
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| Denhardt Solution (100X) |
| 10 g Ficoll 400
10 g Polyvinylpyrrolidone
10 g Bovine Serum Albumin (Fraction V)
Bring final volume to 500 ml with ddH2O
Store at -20°C in aliquots
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| SSCP (20X) |
| 2.4 M NaCl
0.3 M Sodium Citrate
0.4 M Sodium Phosphate, pH 6.8
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| 1 M HCl |
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| SSC (20X) |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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| Stripping Solution |
| 0.1% (w/v) SDS
0.1X SSC
0.2 M Tris-HCl, pH 7.5
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| Hybridization Buffer |
| 0.1% (w/v) SDS
5X Denhardt Solution
5X SSCP
200 μg/ml Herring Sperm DNA
Prepare fresh just before use.
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| Neutralization Buffer |
| 1 M Tris-HCl, pH 7.4
1.5 M NaCl
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| Denaturing Solution |
| 1.5 M NaCl
0.5 M NaOH
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| 50 mM HCl |
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Hybond N™ Membrane
SDS
Tris
Ficoll 400
Oligonucleotide
Sodium Phosphate
Hydrochloric Acid
Sodium Citrate
Bovine Serum Albumin (BSA), Fraction V
DNA, Herring Sperm
Sodium Chloride
Polyvinyl Pyrolidone
Sodium Hydroxide
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Cut the gel just in front of the wells, leaving about 1 cm around the left, right and bottom edges beyond the sample lanes.
3. Do not move the Hybond-N™ membrane once it has touched the gel.
4. The membrane can be placed on Whatman 3MM paper, covered with plastic wrap, and stored for months.
5. You can also use a glass pipette to remove some of the air bubbles between the bottle and the filter.
6. If the filter rolls up, turn the bottle around in the oven and the membrane should unwind on its own. Do not tighten the cap too much and make sure that the bottle is preheated to 65°C or else it may break.
7. A base- or boiled-denatured probe is fine. It is also OK to use the prehybridization buffer for the hybridization. To base denature a probe, dilute the probe to 200 μl with ddH2O. Add 20 μl of 1 M NaOH. Incubate for 5 min at room temperature and then for 5 min on ice. Add 20 μl 1M HCl and 20 μl of 1 M Tris-Cl, pH 8.0. Add to pre-hybridization buffer. To denature a probe by boiling, place tube containing the probe at 100°C for 5 min. Incubate probe on ice for 5 min. Add to pre-hybridization buffer.
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