Preparation of λ phage proto-species assay by plate lysis and elution
The preparation of λ phage progenitors derived from a single phage spot is usually done by two methods: (i) plate lysis, which is a method of phage proliferation in bacteria grown on top agar or agarose; and (ii) small-volume liquid culture, which is a method of phage proliferation with bacteria grown in liquid medium. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Preparation of λ phage protospecies experiments by plate lysis and elution
Principle
Preparation of λ phage progenitors derived from individual phage spots is usually done by two methods: (i) plate lysis, a method of phage proliferation in bacteria grown on top agar or agarose, and (ii) small volume liquid culture, a method of phage proliferation with bacteria grown in liquid media.
Materials and Instruments
λ Phage Progenitor species Escherichia coli spread plate bacteria Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform SM
LB or NZCYM Agar plates Sorvall SS-34 or equivalent Water bath Screw or bayonet polypropylene tubes
1. Buffers and solutions
Chloroform, SM.
2. Culture medium
LB or NZCYM agar plates 
LB or NZCYM top agarose (0.7%)
3. Centrifuge and rotor
Sorvall SS-34 or equivalent
4. Specialized equipment
Heating unit or pre-set 47°C water bath
Screw or bayonet polypropylene tubes (13 mm X 100 mm or larger)
5. Carriers and strains
λ Phage Progenitor
E. coli spread plate bacteria
II. METHODS
1. Preparation of plate spreading infection cultures:
For a 10 cm diameter petri dish, take 105 pfu of phage (usually about 1/10 of a phage spot resuspension or 1/100 of a macrophage spot resuspension) and mix with 0.1 ml of plate-laying bacteria.
For a 15 cm diameter petri dish, take 2X105 pfu of phage and mix with 0.2 ml of plate-laying bacteria.
At least one control tube containing uninfected cells should be set up, and both infected cultures and controls should be incubated at 37℃ for 20 min to adsorb the virus onto the cells.
If λ phage progenitors are prepared that do not grow easily, 106 pfu of phage should be inoculated for every 0.1 ml of plate-spread bacteria.
2. Add 3 ml of melted 47°C agarose (10 cm plates) or 7 ml (15 cm plates) to the first tube of infected cells, mix by tapping or vortexing for a few seconds, and pour immediately into the center of the labeled agar plate. Try to avoid air bubbles. Rotate the plate to ensure even distribution of bacteria and top agarose. Repeat this step until everything in each tube is transferred to the individual plates.
3. Place the plate in the positive position at 37 °C for about 12-16 h.
The reason the plates are not inverted during incubation is to encourage the formation of water droplets on the surface of the flat dish, which makes it easier for the phage to spread.
At the time of collection, the phage spots should be in contact with each other, and the only visible sign of bacterial growth is the thin, transparent marginal band that marks the site of binding of neighboring phage spots. Plates containing uninfected cells should form a smooth patch of bacterial moss.
4. Remove the plates from the incubator, add SM (5 ml for 10 ml plates, 10 ml for 15 ml plates) and shake for several hours at 4°C on a shaker.
5. Using separate Pasteur pipettes, transfer as much SM as possible from each plate to a screw or bayoneted sterile polypropylene test tube.
6. Add another 1 ml of fresh SM to each plate, shake the liquid gently, then leave the plate at an angle for 15 min so that all the liquid is concentrated in one place, then aspirate the SM and mix it with the first collection and discard the plate.
7. Add 0.1 ml of chloroform to each tube containing SM, gently vortex and shake, and then remove cell debris by centrifugation at 4000 g (5800 r/min for Sorvall SS-34) at 4°C for 10 min.
8. Transfer the supernatant to new polypropylene tubes and add one drop of chloroform to each tube. The resulting phage plate culture stock was stored at 4℃.
9. Determine the concentration of infectious virus particles in each stock by phage spot analysis as described in "Plate culture of λ phage". 
