Preparation of λ phage proto-species experiments with small quantities of liquid cultures

Summary

High-titer λ phage progenitors can be prepared from small quantities of liquid cultures. This method was first used by Leder et al. (1977). In general, it yields a lower and less stable yield of λ phage than plate lysis, in addition to a larger initial inoculum of phage. However, λ phage progeny prepared in liquid cultures will be clearer and also free of sulfated polysaccharides that contaminate plate lysates. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" Translated by Huang Peitang et al.

Operation method

Experiments on the preparation of λ phage proto-species from small quantities of liquid cultures

Principle

High-titer λ phage progenitors can be prepared from small quantities of liquid cultures. This method was first used by Leder et al. (1977). In general, it yields a lower and less stable yield of λ phage than plate lysis, in addition to a larger initial inoculum of phage. However, λ phage progeny prepared in liquid cultures will be clearer and free of sulfated polysaccharides that contaminate plate lysates.

Materials and Instruments

λ Phage Progenitor
Chloroform SM
LB or NZCYM agar plates Sorvall SS-34 or equivalent 30°C incubator Polypropylene culture tubes

Move

I. Materials

1. Buffers and solutions

Chloroform, SM.

2. Culture medium

LB or NZCYM medium pre-warmed to 37 °C.

3. centrifuge and rotor

Sorvall SS-34 or equivalent.

4. Specialized equipment

30°C incubator

Polypropylene tubes (17 mm X 100 mm)

5. Carriers and strains

λ Phage Progenitor

II. METHODS

1. Inoculate single colonies of suitable E. coli strains into 5 ml of NZCYM or LB medium and incubate overnight at 30℃ with vigorous shaking.

2. Transfer 0.1 ml of fresh bacterial overnight culture (prepared in step 1) to a sterile 17X100 mm polypropylene tube with a loose-fitting cap, and then add 50-100 μl of SM containing approximately ¹⁰⁶ pfu of λ phage for infection.

3. Place the infected culture at 37℃ for 20 min to allow phage particles to adsorb onto the bacteria.

4. Add 4 ml of pre-warmed NZCYM or LB medium and incubate with vigorous shaking until lysis occurs (usually 8~12 h at 37℃).

If lysis has not occurred or is incomplete after 12 hours of incubation, add an equal volume of pre-warmed NZCYM or LB medium to the culture, and continue to incubate at 37℃ with vigorous shaking for 2~3 hours.

5. After cleavage occurs, add two drops (about 100 μl) of chloroform and continue incubation at 37℃ for 15 min.

6. Centrifuge at 4000 g (5800 r/min Sorvall SS-34 rotor) at 4℃ for 10 min.

7. Recover the supernatant, add 1 drop (about 50 μl) of chloroform, and store the virus stock.

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