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The Laboratory of Andrew Murray at Harvard University
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| This is a detailed protocol on the mechanics of performing Southern blot analysis of DNA using Church Buffer in the hybridization of probe to membrane. Church Buffer is a simpler hybridization buffer due to its minimal components. The conditions for hybridization and washing of the Southern blot are general and should be optimized for detection of the gene of interest. |
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A. Agarose Gel Preparation
1. Run purified restriction-digested yeast DNA on a 0.85% Agarose gel (See Agarose Gel DNA Electrophoresis Protocol) (see Hint #2).
2. Stain gel in 0.5 μg/ml Ethidium Bromide and destain in ddH2O or add Ethidium Bromide to Agarose gel. Photograph the Agarose gel next to a clear or glow-in-the dark ruler on UV box.
3. Depurinate the DNA by soaking the gel with gentle agitation in 0.2 M HCl for 10 min only if the fragments of interest are larger than 15 Kb. .
4. Rinse the gel with ddH2O.
5. Denature the DNA by soaking the gel with gentle agitation in Denaturing Buffer for 15 min. Pour off solution, add more Denaturing Solution, and soak another 15 min.
6. Rinse gel with ddH2O.
7. Neutralize the gel by soaking gel with gentle agitation for 30 to 45 min in Gel Neutralizing Buffer, changing the buffer several times.
B. Transfer
1. Fill the bottom of an oblong baking dish with about 1.5 to 3 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.
2. 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 (see Hint #3).
3. Using gloved hands and forceps to handle the membrane, cut a piece of Nitrocellulose Membrane or Nylon Membrane the size of the gel. The membrane can be smaller than the gel; be sure to include all parts of the gel encompassing the expected bands. Trim the lower left-hand corner of the membrane.
4. Wet the membrane in ddH2O and then soak it in 10X SSC for about 10 min.
5. Place the membrane on the gel. Smooth out any bubbles between the gel and the membrane using a glass rod or pipette. 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.
6. 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 (see Hint #4).
7. 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.
8. 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.
9. Place the two dry pieces of Whatman 3MM filter papers on the stack.
10. 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 a 500 g weight on top of the glass plate.
11. Place Saran 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 Saran wrap so that the reservoirs of buffer are covered and do not evaporate during the course of the transfer.
12. Transfer overnight at room temperature.
C. Hybridization
1. Disassemble the transfer setup. Remove the paper towels and the Whatman 3MM filter papers above the gel. Wearing gloves, turn over the gel and the filter and lay them gel side up on a dry sheet of Whatman 3MM paper. Mark the positions of the gel wells on the filter with a soft lead pencil or a ballpoint pen.
2. Peel the gel from the membrane and discard the gel. Soak the membrane in 6X SSC for 5 min at room temperature to remove any excess Agarose.
3. Drain the membrane and dry it on a piece of Whatman 3MM paper for at least 30 min.
4. Covalently link the DNA to the membrane with a UV-cross linker. The side of the membrane carrying the DNA must face the UV source. Alternatively, sandwich the membrane between 2 pieces of Whatman 3MM paper and bake the membrane for 90 min at 80°C in a vacuum oven to fix the DNA onto the membrane. If a Nitrocellulose membrane is used, you must bake the membrane.
5. Wet the filter with 6X SSC.
6. Pre-hybridize the membrane in Church Buffer for 30 min at 65°C.
7. Label the DNA probe (See appropriate Protocols for Labeling a DNA probe).
8. If the probe is double-stranded, boil 100 μl of probe for 5 min to denature the DNA and immediate transfer it to an ice bath to snap cool for 5 min. Dilute the probe to 1 ml in Church Buffer and add it to the pre-hybridization Church Buffer and filter or carefully add the undiluted probe to the solution, being careful not to add the probe directly onto the membrane (see Hints #5 and #6).
9. Hybridize overnight at 65°C.
10. Remove the hybridization solution and add 100 to 300 ml Wash Buffer 1. Incubate for 5 min at 65°C. Replace with fresh Wash Buffer 1 and incubate for 15 min at 65°C.
11. Remove Wash Buffer 1. Replace it with 100 to 300 ml Wash Buffer 2, and incubate for 30 min at 65°C, with a change of buffer after 15 min.
12. Remove Wash Buffer 2. Replace it with 100 to 300 ml Wash Buffer 3 and incubate for 30 min at 65°C, with a change of buffer after 15 min.
13. Wrap the moist (but not dripping wet) membrane in saran wrap. Do not let the membrane dry out if it is to be either reprobed or washed further later.
14. Employ the proper detection system for the probe used (autoradiography, fluorescence, chemiluminescence, etc.)
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| Church Buffer |
| 0.17% (v/v) Phosphoric Acid
1% (w/v) Bovine Serum Albumin
1 mM EDTA
7% (w/v) SDS
35.5 g/liter Sodium Phosphate Dibasic (Na2HPO4)
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| SSC (10X) |
| pH 7.2
0.15 M Sodium Citrate
1.5 M NaCl
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| Gel Neutralizing Buffer |
| 1 M Tris, pH 7.4
1.5 M NaCl
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| Denaturing Buffer |
| 1.5 M NaCl
0.5 M NaOH
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| 0.2 M HCl |
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| 0.5 μg/ml Ethidium Bromide |
| CAUTION! see Hint #1
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| Wash Buffer 3 |
| 0.1% (w/v) SDS
Pre-warmed to 65°C
0.1X SSC
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| Wash Buffer 2 |
| 0.1% (w/v) SDS
Pre-warmed to 65°C
0.5X SSC
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| Wash Buffer 1 |
| 0.1% (w/v) SDS
Pre-warmed to 65°C
2X SSC
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Ethidium Bromide
Tris
Sodium Phosphate, Dibasic
Sodium Chloride
Phosphoric Acid
Bovine Serum Albumin (BSA)
EDTA
SDS
Sodium Citrate
Hydrochloric Acid
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1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Use 5 to 10 μl of Winston prep DNA (see Protocol for Winston Prep DNA).
3. Flipping the gel so that the Nitrocellulose Membrane is in contact with the smooth bottom surface of the gel prevents unsightly background swirls on the developed film. These are caused by the top surface of the poured gel.
4. It is important that the gel or the underlying Whatman 3MM support does not come in direct contact with the upper layers of Whatman paper or paper towels. This will short circuit the transfer and lead to uneven and inefficient transfer of DNA.
5. Hybridize in a volume of at least 0.2 ml buffer per cm2 of membrane.
6. It is not necessary to change the pre-hybridization Church Buffer before starting the hybridization.
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