| Contributor: |
The University of Maryland Baltimore County Applied Molecular Biology Program
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1. Use the appropriate restriction digested genomic DNA to generate specific fragment sizes for the detection of the target DNA locus.
2. Prepare a 0.8 to 1.0% agarose gel containing 1X TAE Buffer. Ethidium Bromide can be added to the gel to a final concentration of 0.2 μg/ml. Use 1X TAE Buffer as running buffer in the gel box.
3. Add one-tenth volume 10X Loading Buffer and load the samples and DNA markers on the agarose gel.
4. Electrophorese at 4V per cm until the Bromophenol Blue dye front indicates that the sample has run for a sufficient distance.
5. Following electrophoresis, visualize the DNA by UV transillumination.
6. Photograph gel with a ruler.
7. Incubate gel in Depurination Solution for 10 min at room temperature with gentle agitation. This step is only necessary if the digested target DNA is greater than 10 kb.
8. Remove gel from Depurination Solution and wash with ddH2O.
9. Incubate gel in Denaturation Solution for 25 min at room temperature with gentle agitation.
10. Remove the gel from the Denaturation Solution and wash with ddH2O.
11. Incubate the gel in Neutralization Solution for 30 min at room temperature with gentle agitation (see Hint #2).
12. Remove the gel from the Neutralization Solution and wash with ddH2O.
13. Fill the bottom of an oblong baking dish with about 1.5 cm to 2.5 cm of 10X SSC and lay a glass plate across the width of the dish. Drape a piece of Whatman 3MM paper pre-wetted with 10X SSC across the glass plate so that the ends of the Whatman 3MM paper extend beyond the edges of the glass plate and are submerged in the buffer below. Smooth out all the air bubbles between the paper and the glass plate with a glass rod or glass pipette.
14. Cut the lower left-hand corner of the neutralized gel for later orientation purposes. Flip the gel and place it on the moist Whatman paper so that the top surface of the gel is now against the Whatman paper.
15. Using gloved hands and forceps to handle the membrane, cut a piece of Nitrocellulose Membrane or Nylon Membrane the size of the gel. Trim the lower left-hand corner of the membrane(see Hint #3).
16. Wet the membrane in ddH2O and then soak it in 10X SSC for about 10 min.
17. Place the membrane on the gel (see Hint #4). Smooth out any bubbles between the gel and the membrane using a
glass rod or pipette.
18. Lay down strips of Parafilm along each of the four edges of the membrane to cover any exposed parts of the gel. Overlap the Parafilm on top of the membrane by several mm.
19. Cut 3 pieces of Whatman 3MM paper to the exact same size as the membrane and wet one of them with 10X SSC. Blot it on another (scrap) piece of Whatman 3MM paper.
20. Lay the damp filter paper piece on top of the Nitrocellulose or Nylon Membrane. Smooth out any air bubbles between the paper and the membrane.
21. Place the two dry pieces of Whatman 3MM filter papers on the stack.
22. Place a 5 to 8 cm thick layer of paper towels on top of the filter papers. Then place a glass plate on top of the paper towels and add about a 500 g weight on top of the glass plate.
23. Place plastic wrap on top of the Whatman paper-covered glass plate support so that none of the paper towels or filters in the upper stack touch the lower wetted Whatman paper underneath the gel. Extend the plastic wrap so that the reservoirs of buffer are covered and do not evaporate during the course of the transfer.
24. Allow the DNA to transfer overnight.
25. Disassemble the transfer apparatus and mark the membrane appropriately for alignment.
26. Fix the DNA to the membrane by UV crosslinking (nylon membrane) or by baking for 2 hr at 80°C in a vacuum oven (nitrocellulose membrane).
27. See Protocol on Southern Blotting for hybridization of probe to the membrane.
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| TAE Buffer (50X) |
| 37.2 g Disodium EDTA
57.1 ml Glacial Acetic Acid
Add ddH2O to 1 liter
242 g Tris base
pH 8.5
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| SSC (20X) |
| 3 M NaCl
0.3 M Sodium Citrate
pH 7.0
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| Neutralization Solution |
| 0.5 M Tris-HCl
Adjust pH to 7.5
1.5 M NaCl
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| Denaturation Solution |
| 1.5 M NaCl
0.5 M NaOH
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| Depurination Solution |
| 250 mM HCl
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| Loading Buffer (10X) |
| 0.1% Xylene Cyanol
0.1% Bromophenol Blue
50% (v/v) Glycerol
0.1M EDTA, pH 8.0
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| 20 μg/ml Ethidium Bromide |
| (CAUTION! see Hint #1)
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Tris
EDTA
Sodium Citrate
Glycerol
Nylon Membrane
Sodium Chloride
Disodium EDTA
Sodium Hydroxide
Ethidium Bromide
Agarose
Glacial Acetic Acid
Xylene Cyanol FF
Bromophenol Blue
Hydrochloric Acid
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for
proper handling instructions.
2. When using nitrocellulose membranes the neutralization incubation should be extended to 45 min.
3. The membrane can be smaller than the gel; be sure to include all parts of the gel encompassing the expected bands.
4. Do not move the membrane once it has been placed on the gel; shifting the membrane will lead to smeared background bands caused by the transfer of DNA during the short time the membrane was in contact with the gel.
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