The signal intensity obtained by Southern hybridization depends on several factors, including the ratio of immobilized DNA complementary to the probe, the size and specific activity of the probe, and the amount of genomic DNA transferred to the membrane. Under optimal conditions, this method is sensitive enough to detect <0.1 pg of DNA complementary to a 32P-labeled, highly specific (>109 cpm/μg) probe.Source: "A Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
Southern hybridization of radiolabeled probes with nucleic acids immobilized on membranes
Principle
The signal intensity obtained by Southern hybridization depends on several factors, including the ratio of immobilized DNA complementary to the probe, the size and specific activity of the probe, and the amount of genomic DNA transferred to the membrane. Under optimal conditions, this method is sensitive enough to detect <0.1 pg of DNA complementary to a 32P-labeled, highly specific (>109 cpm/μg) probe.
Materials and Instruments
DNA immobilized on membrane Sterile aqueous poly (A) RNA DNA or RNA probe Salmon sperm DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Phosphate-SDS wash Prehybridization Hybridization solution Aqueous buffer solution for hybridization Formamide buffer solution for hybridization
Radioactive ink-labeled self-adhesive labels or phosphorescent self-adhesive labels Hybridization vials Pre-heated incubator or commercially available hybridization device Incubator or water-bath oscillator Boiling water bath
1. Buffers and solutions
Phosphate-SDS Wash 1 (40 mmol/L Na3PO4 (pH 7.2), 1 mmol/L EDTA (pH 8.0), 5% (m/V) SDS, 0.5 (m/V) bovine serum albumin component V)
Phosphate-SDS Wash 2 (40 mmol/L Na3PO4 (pH 7.2), 1 mmol/L EDTA (pH 8.0), 5% (m/V) SDS)
Pre-Hybridization/Hybridization Solution
Aqueous buffer solution for hybridization (6X SSC (or 6X SSPE), 5X Denbardts reagent, 0.5% (m/V) SDS, 1 μg/ml poly (A), 100 μg/ml salmon sperm DNA, 50% (V/V) formamide)
Formamide buffer solution for hybridization (0.5 mol/L Na3PO4 (pH 7.2), 1 mmol/L EDTA ( pH 8.0), 7% (m/V) SDS, 1% (m/V) bovine serum albumin)
Na3PO4 (1 mol/L, pH 7.2)
2. Nucleic acids and oligonucleic acids
DNA immobilized on the membrane
Poly (A) RNA in sterile water (10 mg/ml)
DNA or RNA probe
Salmon sperm DNA (approx. 10 mg/ml)
3. Specialized equipment
Radioactive ink labeled self-adhesive labels or phosphorescent self-adhesive labels
Hybridization bottles
Incubator or commercially available hybridization device pre-conditioned to the appropriate temperature
Incubator or water bath shaker pre-conditioned to 65°C (for hybridization in phosphate-SDS buffer)
Boiling water bath
ii. Methods
1. Float the membrane containing the target DNA in a dish containing 6X SSC (or 6X SSPE) until the membrane is completely wetted from the bottom up. Submerge the membrane in the solution for 2 min.
2. Pre-hybridization is performed by one of the following methods
(1) Hybridization in a heat-sealed bag
① Stuff the membrane into a heat-sealed bag (e.g., Sears Seal-A-Meal or equivalent device) and add 0.2 ml per square centimeter to the prehybridization solution. Squeeze as much air out of the bag as possible.
② Seal the open end of the bag twice with a heat sealer. Gently squeeze the bag to check the strength and integrity of the seal. Place the bag in a water bath at the appropriate temperature (68°C for aqueous solvent hybridization solution; 42°C for hybridization solution containing 50% formamide; 65°C for phosphoric acid-SDS hybridization solution).
(2) Hybridization in Hybridization Vials
① Gently roll the moistened membrane into a cylindrical shape and place it into the hybridization vial together with the plastic mesh provided by the manufacturer. Add prehybridization solution at 0.1 ml per square centimeter of membrane. Tighten the cap.
② Place the hybridization tubes in a hybridization oven preheated to the appropriate temperature (68°C for aqueous solvent hybridization solution; 42°C for hybridization solution containing 50% formamide; 65°C for phosphoric acid-SDS hybridization solution).
(3) Hybridization in plastic containers
① Place the wetted membrane in a plastic (e.g., Tupperware) container and add the prehybridization solution at 0.2 ml per square centimeter of membrane.
② Seal the box with the lid and place the box on an oscillating platform in an air incubator preset to the appropriate temperature (68°C for aqueous solvent hybridization solution; 42°C for hybridization solution containing 50% formamide; 65°C for phosphoric acid-SDS hybridization solution).
3. If the radiolabeled probe is double-stranded DNA, denature the probe by heating at 100°C for 5 min and rapidly cool the probe in an ice-water bath.
4. To hybridize the probe to a membrane containing genomic DNA, proceed as follows
(1) Hybridization in a heat-sealed bag
① Quickly remove the plastic bag containing the membrane from the water bath. Open the bag by cutting one corner with scissors and pour out the prehybridization solution.
② Add the cross-sexed probe to an appropriate amount of fresh pre-hybridization solution and transfer the solution into the bag. Squeeze as much air out of the bag as possible.
③ Reseal the bag with a heat sealer; leave as few air bubbles in the bag as possible. To avoid radioactive contamination of the water bath, seal the resealed bag in another uncontaminated bag. Immerse the bag in a water bath at a temperature appropriate for the desired hybridization stage.
(2) Hybridization in Hybridization Vials
① Pour the prehybridization solution out of the hybridization vial and add fresh hybridization solution containing the probe.
② Seal the bottle and put it back into the hybridization oven. Place the disk at the appropriate temperature.
(3) Hybridization in a plastic container
① Transfer the membrane from the container to a sealed bag or hybridization bottle.
② Quickly dispose of them as described above.
5. After hybridization, wash the membrane.
(1) Hybridization in a heat-sealed bag
① Wearing gloves, remove the bag from the water bath, remove the outer bag, and quickly cut off one corner of the inner bag. Pour the hybridization solution into a vat of waste solution for disposal of radioactive contamination, and then cut the bag open along three sides.
② Remove the membrane and quickly immerse it in a dish containing several hundred milliliters of 2X SSC and 0.5% SDS (i.e., approximately 1 milliliter per square centimeter of membrane), and gently oscillate the dish on a slow rotating platform at room temperature.
(2) Hybridization in hybridization flasks
① Remove the membrane from the hybridization flask and dry the excess hybridization solution by clamping the corner of the membrane that is exposed to the mouth of the flask.
② Submerge the membrane into a dish containing several hundred milliliters of 2X SSC and 0.5% SDS (i.e., about 1 ml per square centimeter of membrane at room temperature) and gently shake the dish on a slow rotating platform.
6. 5 min later, pour the first wash into the waste tank for radioactive contamination and add several hundred milliliters of 2X SSC and 0.1 % SDS to the dish. leave at room temperature for 15 min and shake gently several times.
If the hybridization was performed in phosphate-SDS buffer, the membrane is immersed in Phosphate-SDS Wash 2 at 65 °C eight times for 5 min each time, and then proceed directly to step 9.
7. Replace the soaking solution with several hundred milliliters of fresh 0.1X SSC containing 0.1% SDS. Allow to stand at 65°C for 30 min to 4 h with gentle shaking.
8. Wash the membrane with 0.1X SSC at room temperature.
9. Place the membrane on a stack of paper towels to remove most of the liquid. Place the wet membrane on a sheet of Saran wrap. Apply adhesive dot labels labeled with radioactive ink (or phosphorescent dots) to several asymmetrical locations on the Saran wrap. These markers can be radiographically self-developed with the film. Cover the label with Scotch film. This prevents contamination of the developer clip or contamination of the sensitizing screen with radioactive ink.
Alternatively, the film can be dried in air and glued to a piece of 3 MM filter paper with water-soluble glue.
10. Cover the film with Saran wrap and place it inside the intensifier screen at -70°C and expose it to x-rays for 16-24 hours to obtain a radiographically autoradiographic image. 
