Phage DNA hybridization experiments on filter membranes
Filter membranes carrying immobilized DNA derived from phage spots can be screened by in situ hybridization with 32P-labeled probes. The technique is robust, highly specific and sensitive, and can identify a single recombinant from thousands of phage spots. This experiment is from "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Hybridization experiments of phage DNA on filter membranes
Principle
Filter membranes carrying immobilized DNA derived from phage spots can be screened by in situ hybridization with 32P-labeled probes. The technique is robust, highly specific and sensitive, and can identify a single recombinant from thousands of phage spots.
Materials and Instruments
Phage DNA radiolabeling probe Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform Pre-Hybridization Solution SM SSPE
Boiling water bath Glass (Pyrex ) Toasted flatware or other hybridization vessels Glue Incubator Radioactive ink Whatman 3 MM paper
1. Buffers and solutions
Chloroform
Pre-hybridization solution
SM
2X SSPE
Wash 1 (2X SSC, 0.1% (m/V) SDS)
Wash 2 (1X SSC, 0.1% (m/V) SDS)
Wash 3 (0.1X SSC, 0.1% (m/V) SDS)
2. Nucleic acids and oligonucleotides
Phage DNA immobilized on membrane
Radiolabeled probes
3. Specialized equipment
Boiling water bath (for denaturation of double-stranded probes)
Glass (Pyrex ) baking pans or other hybridization vessels 
Glue, water-soluble (e.g., UHU Stic from FaberCastell)
Incubator pre-set at the appropriate hybridization temperature
Radioactive ink
Whatman 3 MM paper
II. Methods
1. If the membrane is dry, float the baked or cross-linked membrane on the 2X SSPE surface until it is completely wetted from the bottom up, then submerge the membrane for 5 min.
2. Transfer the membranes to Pyrex dishes or other hybridization vessels containing prehybridization solution at 3 ml of prehybridization solution per 82 mm membrane or 5 ml of prehybridization solution per 132 mm membrane. The membranes are gently shaken in a shaker at a suitable temperature (i.e., 68°C in aqueous solution or 42°C in 50% formamide solution) for 1 to 2 h or longer.
3. Denature the 32P-labeled double-stranded probe by heating at 100°C for 5 min and then rapidly cool it in an ice-water bath, while the single-stranded probe does not need to be denatured. 
4. Add the denatured probes to the membrane-impregnated prehybridization solution and incubate for 12-16 h at a suitable temperature.
5. When hybridization is complete, quickly remove the membrane from the hybridization solution and immediately immerse it in a large volume (300-500 ml) of Wash Solution 1 at room temperature. Shake the membrane gently and turn it over at least once during the rinsing process. 5 min later, transfer it to fresh wash solution and continue to shake it gently. Repeat the rinsing process twice more.
6. Rinse the membrane twice in 300-500 ml of Wash 2 at 68 °C for 1-1.5 h.
7. Place the membrane on Whatman 3 MM filter paper or paper towel and air dry at room temperature. Apply water-soluble glue to the back of the membrane and arrange it on a clean, dry, flat piece of 3 MM filter paper (numbered side up) and press the membrane firmly so that it adheres to the paper and does not move. Several asymmetric spots on the 3 MM filter paper were selected and labeled with drops of radioactive ink or chemiluminescent markers. These markers were used to align the radioautography with the filter membrane. Cover the membrane and markers with Saran's Wrap/Cling Film. Tape the film to the back of the 3 MM filter paper and straighten the film on the filter paper to prevent wrinkling.
8. Expose the membrane to X-rays (Kodak XAR-2, XAR-5, or equivalent) with a sensitizing screen at -70°C for 12 to 16 hours.
9. The x-ray film is developed, aligned with the membrane according to the radioactive ink or chemiluminescent markings, and the asymmetric points corresponding to the numbered membranes are marked on the x-ray film at the appropriate locations with a non-radioactive red fiber-tip pen.
10. Positive clones are found by comparison with the orientation markings on the agar plate.
11. Pick the positive clones and store in 1 ml SM containing 1 drop (50 μl ) of chloroform.
12. To purify the hybridization-positive phage spots, the phage fraction recovered from the snap agar block (usually 50 μl 10-2 dilution) is spread on a plate for incubation, followed by a new round of hybridization screening.