Purification experiments of phage clones
Purification experiments of phage clones
Phage is a general term for bacteriophages that infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. As a type of virus, phages have some characteristics unique to viruses: they are individually tiny; they do not have a complete cellular structure; and they contain only a single nucleic acid. The phage genome contains many genes, but all known phages utilize bacterial ribosomes, various factors required for protein synthesis, various amino acids and energy generating systems in bacterial cells to achieve their own growth and proliferation.
Operation method
basic program
Materials and Instruments
Phage Move 1. Pour 3 ml of 0.7% top agarose containing 200 μl of host bacteria on a 82 mm diameter LB flat pole and leave for 10 min.2. Determine the position of the positive phage spot on the original plate by radiating the hybridization spot on the autoradiographic film and gently inserting a toothpick into the spot. For more product details, please visit Aladdin Scientific website.
LB top agarose Chloroform SM MgSO4
Incubator Toothpick Cellulose nitrate membrane
3. then inoculate into the top layer of agarose of the secondary plate prepared in step 1. this toothpick is inserted into the plate 30 to 40 times in succession.4. or insert a pipette into the site of a positive phage spot and take a small piece of ring agarose and transfer it into 1 ml of SM.
5. Add 1 drop of chloroform, leave for 1-2 h, titrate, and prepare 3-6 plates, the number of phage spots per plate should be less than 500.
6. Place the secondary plate at 37°C for overnight incubation and photocopy the phage spots onto nitrocellulose filter membrane.
7. Then process, hybridize, wash the membrane and expose sequentially, and then find out the corresponding positive phage spots on the secondary plates.
9. For each clone, a toothpick was inserted into the phage spot with the strongest hybridization signal and placed in 1 ml SM for 5 min.
10. 1 μl of phage reservoir solution was placed. 1 μl and 10 μl of 1:100 dilution were spread on tertiary plates.
11. Screen the phage spots on the tertiary plate as above.
12. Repeat this purification process until pure phage spots are obtained and store the pure phage in SM.