Adapted from M. Marston, Roger Williams University and Jay T. Lennon, Brown University

Supplies:

  • SN plates
  • Washed agar
  • 75% Seawater
  • 1.5 ml eppendorf tubes (sterile)
  • Culture Tubes (sterile)
  • Vortex
  • Centrifuge
  • Chloroform
  • Synechococcus WH 7803
  • 24-well microtiter plates

Pouring plates

1. Mix 0.6g washed agar with 100ml 75% seawater and autoclave. After autoclaving let cool in 55-65 oC waterbath. Then add salts and cyanometals. Mix and pour into Petri plates until a layer covers the bottom. Leave still to harden and then store upside down in incubator or in a 4C fridge.

2. Make a solution of 0.35% washed agar and SN media. May autoclave or microwave. Cool and store at 65C in a water bath. (Can be done a day or two in advance)

3. Turn on 37 oC heat block and put culture tubes in heatblock to cool agar down. Add 1.5mL of soft agar to sterile test tubes and place on 37C heat block.

4. Make phage dilutions using SN media

  • for (-2) (or another appropriate dilution)add 5uL of stock lysates + 495uL of SN
  • for (-4) (another appropriate dilution)add 5uL of (-2) dilution + 495uL of SN

5. Spin down (1.5 ml * # of lysates) dense culture being purified + two control plates (4500rpm, 10 minutes). Leave (0.2 ml * # of lysates being purified + 2 control plates) liquid to resuspend pellet in.

6. Take out testtube from heat block.

7. Add 40uL of lysate dilution (see number 4) dilution to test tube with soft agar.

8. Add 200uL of WH7803 concentrated cells to test tube. (cells more sensitive to heat is why they are added second)

9. Vortex test tube and pour onto hard agar plates. Swirl and cover. (try to minimize the number of bubbles, these could be mistaken for plaques)

10. Let agar set for about 30 minutes and invert plates, place in ziplock bags

11. Place in a light incubator.

12. Check for plaques after 3 days of purification and from then on every day. If there are plaques on a plate:

13. Add 200uL of cells and 1mL of SN media to a 24 well plate.

Label wells with names of lysates that generated plaques, dilution and date.

14. Pick plaques with a sterile pipet tip or toothpick and transfer in the right well on a 24-well plate.

15. Place in 25C incubator for 3-8 days until well clears.

16. Remove content of clear wells and place in microfuge tube.

17. Add 11uL of chloroform and invert by hand 10 times

18. Centrifuge for 12 minutes at 4,000 rpm.

19. Place in clear, labeled cryogenic vial and store in 4C. (PP name of lysate, dilution and date)