M13 Phage liquid culture
M13 Phage progenitors are usually grown in liquid culture, where infected cells do not lyse but grow slowly to form a dilute suspension. Inoculation is almost always with a suspension of phage particles obtained from a freshly picked phage spot or a single phage spot. Infected cells contain more than 200 copies of double-stranded RF DNA and secrete hundreds of phage particles per generation. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
M13 phage liquid culture
Principle
M13 Phage progenitors are usually grown in liquid culture, where infected cells do not lyse but grow slowly to form a dilute suspension. Inoculation is almost always with a suspension of phage particles obtained from a freshly picked phage spot or a single phage spot. Infected cells contain more than 200 copies of double-stranded RF DNA and secrete hundreds of phage particles per generation.
Materials and Instruments
M13 Plate phage E. coli F' strain Move I. Materials For more product details, please visit Aladdin Scientific website.
LB Agar Flat Wrench M9 Basic Medium Plate LB or YT Medium Sterile toothpick or inoculation needle or glass capillary tube
1. Culture medium
LB medium containing tetracycline or kanamycin or Kafu M9 basic medium.
LB or YT medium
2 X YT medium with 5 mmol/L MgCl2
2. Specialized equipment
Sterile toothpick or inoculation needle or glass capillary tube (50 μl )
3. Vectors and strains
M13 phage plaques spread on agar or agarose plates (method for preparing phage plaques in the upper layer of agar or agarose)
E. coli F' strain, grown on agar plates with good clonal isolation. (Take primary cultures of appropriate strains and line them on M9 basic agar plates, or for antibiotic resistant strains, on LB plates containing appropriate antibiotics, and incubate at 37°C for 24-36 h. The single-stranded DNA of the M13 phage, if it is intended to be used as an oligonucleotide-mediated mutation, should be proliferated in E. coli containing mutations in the dut and ung genes.)
II. Methods
1. Take a newly grown single colony of E. coli with the F' plasmid and inoculate it with 5 ml of enrichment medium (for antibiotic resistant strains, use LB medium with appropriate antibiotics) and shake it gently at 37°C for 12 h. The colony is then incubated for 12 h at 37°C. The colony is then incubated for 12 h at 37°C.
2. Take 0.1 ml of culture and inoculate 5 ml of 2 X YT medium containing 5 mmol/L MgCl2, incubate at 37℃ with vigorous shaking for 2 hours.
3. Dilute 5 ml of culture with 45 ml of 2 X YT medium containing 5 mmol/L MgCl2, and divide each 1 ml into sterile test tubes (13 X 100 mm or 17 X 100 mm) according to the number of phage plaques to be proliferated, and divide another 2 tubes as negative and positive controls for phage growth. Set these tubes aside and use them in step 7.
4. Dispense 1 ml of YT or LB in sterile tubes (13 X 100 mm or 17 X 100 mm), the number of tubes according to the number of phage plaques to be colonized, and 2 additional tubes as negative and positive controls for phage growth.
5. Dip a sterile inoculation needle into the surface of the phage plaque and rinse it in YT or LB medium to prepare a diluted suspension of M13 phage. Pick a blue phage spot as a positive control for phage growth, and pick an E. coli layer without phage spot on the plate as a negative control.
6. Leave the suspension at room temperature for 1~2 h to allow phage particles to diffuse out of the agar.
7. Take 0.1 ml of phage suspension (Step 6) and infect 1 ml of E. coli culture (Step 3) for isolation of viral DNA. incubate at 37℃ with gentle shaking for 5 h. 
8. Transfer the culture solution to a sterile microcentrifuge tube and centrifuge at maximum speed for 5 min at room temperature without disturbing the bacterial sediment, transfer the supernatant to a new microcentrifuge tube.
9. Place 0.1 ml of supernatant in a sterile microcentrifuge tube.
10. The remaining 1 ml of culture supernatant is used to prepare phage single-stranded DNA, and the bacterial precipitate is used to prepare double-stranded RF DNA.