Phage DNA transfer experiments from phage spots to filter membranes
A method for identifying and isolating specific recombinants from λ phage libraries was established early in the history of molecular cloning by Bemon and Davis (1977). This method, which is still frequently used today, involves mass screening of phage spots by in situ hybridization with 32P-labeled probes. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Phage DNA transfer experiments from phage spots to filter membranes
Principle
A method for identifying and isolating specific recombinants from λ phage libraries was established early in the history of molecular cloning by Bemon and Davis (1977). This method, which is still frequently used, involves mass screening of phage spots by in situ hybridization with 32P-labeled probes.
Materials and Instruments
λ phage library E. coli spread plate bacteria Move I. Materials For more product details, please visit Aladdin Scientific website.
Denaturing solution Neutralizing solution SM+ Gelatin SSPE
LB or NZCYM agar plates LB or NZCYM top agarose Crosslinking equipment Microwave or vacuum oven Needles and syringes Cellulose nitrate or nylon membranes Water baths
1. Buffers and solutions
Denaturing solution
Neutralizing solutions
SM+ Gelatin
2X SSPE
2. Culture medium
LB or NZCYM agar plates
LB or NZCYM top agarose
3. Specialized equipment
Cross-linking equipment (e.g. Stratalinker from Stratgene, GS gene Linker from Bio-Rad),
Microwave oven or vacuum oven pre-set at 80°C.
Needles and syringes (21 gauge)
Nitrocellulose or nylon membrane
Water baths preset at 47°C and 65°C
Waterproof black drawing ink (India ink)
Whatman 3 MM paper
Whatman 3 MM Filter Paper (85 mm diameter)
4. Carriers and strains
λ phage library
E. coli spread plate bacteria
II. METHODS
Preparation of filter membranes and transfer of phage spots
1. Preparation of transfer filter membrane:
(1) Number the dry filter membrane with a soft pencil or ballpoint pen.
(2) Soak the membrane in water for 2 min.
(3) Stack the membranes on top of each other with 85 mm diameter Whatman 3 MM filter paper between them.
(4) Wrap the stack of membranes in aluminum foil and autoclave for 15 min at 15 psi (1.05 kg/cm2 ) in a liquid cycle.
2. Dilute the packaging mixture, phage stock or library with SM+ gelatin. Mix the diluted phage stock with an appropriate amount of freshly prepared plate-spread bacteria. Infected bacterial cultures should be incubated at 37℃ for 20 min. 
3. Add 3 ml or 6.5 ml of melted (47°C) top agar to each portion of infected cells, mix well, and pour into individually numbered 90 mm or 150 mm agar plates.
4. Cover the plate, allow the top layer of agarose to solidify, and incubate inverted at 37°C until phage plaques appear and just begin to touch each other (10-12 h).
5. Leave the plate at 4°C for at least 1 h to allow the top layer of agarose to solidify.
6. Remove the plate from the cooler or refrigerator. Photocopy the phage spots of each plate with the first set of labeled filter films. Place a labeled, dry, circular nitrocellulose or nylon membrane on the surface of the top agarose layer so that it is in direct contact with the phage spots.
7. Mark three or more spots around the perimeter of the filter membrane and inject waterproof black drawing ink with a 21-gauge hypodermic needle by piercing the spots up to the agar below.
8. After 1 to 2 min, remove the filter membrane from each plate in turn using a flat-ended forceps (e.g., Milhpore forceps).
Denaturation of phage DNA on filter membranes
9. Place a piece of Whatman 3 MM filter paper (or equivalent) soaked with denaturing solution in a plastic cafeteria dish or Pyrex flat dish and transfer the filter membrane onto the filter paper, phage spot side up, and leave it there for 1~5 min.
10. Transfer the membrane to a Whatman 3 MM filter paper soaked with neutralizing solution, with the phage spot side facing up, and leave for 5 min.
Fix the phage DNA on the filter membrane.
11. Prepare the filter membrane for immobilization.
Immobilize the DNA on the membrane by microwaving or baking: if microwaving, proceed directly to step 13; if baking in a vacuum oven, transfer the membrane to a 3 mm dry filter paper or a stack of paper towels. Dry the membrane at room temperature for at least 30 min.
To immobilize DNA on the membrane by UV cross-linking: place the membrane on a piece of Whatman 3 MM filter paper soaked in 2X SSPE and place it in the vicinity of the UV cross-linker.
12. If desired, after the first set of membranes has been processed, use the second set of membranes for phage spot photocopying. Ensure that both sets of membranes are placed in the same position on the plate.
13. immobilize the DNA from the phage spots onto the filter membranes
Fixation by microwave treatment: Place the wet filter membrane on a piece of dry Whatman 3 MM filter paper and expose it to the microwave oven at maximum power for 2-3 min.
Fixation by baking: Stack the dry filter membranes (step 10) on top of each other, separate the two membranes with dry Whatman 3 MM filter paper, and bake them in a vacuum oven at 80°C for 1~2 h. The membranes should be fixed in a microwave oven.
Fixation by UV cross-linking: This is done with commercially available equipment, following the manufacturer's instructions.
14. After baking or cross-linking, wrap the dry filter membrane loosely in aluminum foil and store at room temperature. Alternatively, if hybridization is performed within approximately one day, wash the membrane with 0.1X SSC or SSPE and 0.5% SDS at 65°C for 30 min and store moist in a sealed plastic bag. 