λ Mass culture (low multiplicity infection) experiments with phages
λ Mass culture (low multiplicity infection) experiments with phages
Mass preparation of λphage can be obtained by either low-mass infection of bacterial cultures or high-mass infection. Immediately after low magnification infection, the culture is transfected into a large volume culture. Because only a small number of cells in the initial bacterial culture are infected, the uninfected cells in the transferred bacterial culture will divide and grow further in the following hours. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Mass culture (low multiplicity infection) experiments with λ phage
Principle
Large volume preparations of λ phage can be obtained by two methods: low magnification infection of bacterial cultures or high magnification infection. Immediately after low magnification infection, the culture is transfected into a large volume culture. Because only a small number of cells in the initial bacterial culture are infected, the uninfected cells in the transferred bacterial culture will divide and grow further in the following hours.
Materials and Instruments
λ Phage Progenitor Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform SM
NZCYM Sorvall SS-34 or equivalent
1. Buffers and solutions
Chloroform, SM.
2. Culture medium
NZCYM
3. Centrifuge and rotor
Sorvall SS-34 or equivalent.
4. carriers and strains
High titer λ phage progenitor
II. METHODS
1. Inoculate single colonies of suitable E. coli strains into 100 ml NZCYM in 500 ml conical flasks and incubate overnight at 37°C with vigorous shaking.
2. Determine the OD600 value of the culture and calculate the cell concentration with 1OD600=1X109 cells/ml.
3. 4 samples, each containing 1010 cells, were centrifuged at 4000 g (5800 r/min Sorvall SS-34 rotor) for 10 min at room temperature, and the supernatant was removed.
4. Suspend each bacterial precipitate in 3 ml of SM.
5. Add an appropriate number of infectious phage particles and shake to fully disperse the phage throughout the culture.
6. Incubate the infected culture at 37℃ for 20 min with occasional shaking.
7. Add each infected sample to 500 ml of NZCYM (in a 2 L flask) preheated to 37°C and incubate at 37°C with vigorous shaking (300 r/min).
8. Monitor the culture for lysis after 8 hours. Lysis occurs after 8 to 12 h along with bacterial and phage growth. If lysis is observed, go to step 10.
9. If lysis is not apparent after 12 h, take a small sample of the culture and examine the phage growth.
(1) Take two samples of infected culture (1 ml each) into a glass test tube.
(2) Add 1 or 2 drops of chloroform (about 50-100 ml) to each tube and incubate at 37°C for 5-10 min with occasional shaking.
(3) Place the two tubes in the light and compare the appearance of the cultures. If the infection is close to complete but the cells have not yet lysed, the chloroform will cause the cells to rupture, thus changing the cloudy culture to clear. In this case, go to step 10.
10. Add 10 ml of chloroform to each flask and continue incubation for 10 min at 37°C with shaking.
11. Cool the culture to room temperature and subsequently precipitate the phage particles. 