M13 Phage spread plates
M13 Phage spread plates
M13 Phage plaques are formed when a single virus infects a single bacterium. The daughter virus particles infect neighboring bacteria and then produce the next generation of virus particles. The spread of the daughter virus particles is limited when the bacteria are grown on semi-solid media (e.g., containing agar or agarose). This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
M13 Phage spread plates
Principle
M13 Phage plaques are formed when a single virus infects a single bacterium. The spread of daughter virus particles, which infect neighboring bacteria and then give rise to the next generation of virus particles, is limited when the bacteria are grown on semi-solid media (e.g., agar-containing or agarose-containing).
Materials and Instruments
M13 Phage Progenitor Phage Plaque E. coli F' Strain Preparation of Master Cultures Move I. Materials For more product details, please visit Aladdin Scientific website.
IPTG solution X-gal solution
LB Agar Plate M9 Basic Medium Plate LB or YT Medium LB or YT Medium Agar Plate LB or YT Medium Agar Plate LB or YT Medium Agar or Agarose Upper layer of agar or agarose 47°C Heater or water bath Ice bath
1. Buffers and solutions
IPTG solution (20%, m/V)
X-gal solution (2%, m/V)
2. culture medium
LB agar flats containing tetracycline or kanamycin or Garfield's M9 basic medium plates.
LB or YT medium
LB or YT medium agar plates with 5 mmol/L MgCl2
Upper agar or agarose layer of LB or YT medium with 5 mmol/L MgCl2
3. Specialized equipment
47°C heater or water bath
Ice bath
4. Carriers and strains
M13 phage progeny in LB or YT medium or phage plaque suspended in 1 ml LB or YT medium
Master cultures prepared from E. coli F' strains
II.
1. Take the master culture of the strain with F' plasmid and streak it on Garfield M9 basic agar plate or LB plate containing tetracycline (XL1-Blue) or kanamycin (XL1-Blue MR F'Kan). incubate it at 37℃ for 24~36 hours.
2. Prepare bacteria for plate spreading. Pick a single well-isolated single colony on the plate prepared in step 1 and inoculate it into a 20 ml sterile test tube containing 5 ml LB or YT medium, and incubate it on a rotary shaker at 37℃ for 6~8 h. Cool it in an ice bath for 20 min and store it at 4℃. The bacteria can be stored at 4℃ for more than 1 week.
3. Prepare sterile test tubes (13 x 100 mm or 17 x 100 mm) containing 3 ml of melted or YT medium with a top layer of agar or agarose, add 5 mmol/L MgCI2 to the medium, and keep the tubes warm in a heater or a water bath at 47 °C.
4. Label a series of sterile test tubes (13 x 100 mm or 17 x 100 mm) according to the dilution and the amount of original phage (see step 5). Add 100 μl of the phage solution prepared in step 2 to each tube.
5. Make a 10-fold serial dilution ( 10-6 to 10-9 ) of the original phage in LB or YT medium. Add 10 μl or 100 μl of each dilution of phage solution to the test tubes containing the plate-laying bacteria prepared in step 4, and mix the phage and bacteria by gently shaking on a shaker.
Unlike the λ phage, the M13 phage adsorbs bacteria quickly, so it is not necessary to incubate the phage with the plate bacteria before adding the top layer of agar.
6. Add 40 μl of 2% X-gal and 4 μl of 20% IPTG to the tubes containing the upper layer of agar. immediately pour the contents of each tube into a tube containing the infected culture, and mix the culture with the agar/agarose by shaking gently for 3 s on a shaker. The mixture was poured into labeled LB or YT plates containing 5 mmol/L MgCl2 and pre-equilibrated to room temperature. Rotate the plate to ensure uniform distribution of the bacteria and the upper agar layer.
7. Repeat step 6 for each tube containing infected culture prepared in step 5 by adding the upper agar layer containing X-gal and IPTG.
8. Cover the dish and allow to stand at room temperature for 5 min to allow the top agar/agarose to solidify. Wipe any excess condensation from the lids with Kimwipes and place the plates upside down at 37°C.