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1. Prepare liquid media, plates, etc.
- a) (6) 30 mL aliquots LB
- b) (4) 100 mL aliquots LB
- c) 1 L LB bottom agar (20-30 plates)
- d) 300 mL LB top agar
- e) 300 mL buffer NMT (200 mM NaCl, 50 mM MgCl2, 10 mM Tris, pH 7.5-8.0)
- f) 1 N NaOH
2. Streak cells for single colonies and incubate at 37 °C, overnight.
- a) Salmonella typhimurium strain 7136 (suppressor - )
- b) Salmonella typhimurium strain 7155 (suppressor + )
3. Make a 30 mL ON of each strain. Note: At this point, pick from the smeared growth or more than one colony. If you pick a single colony, you may get a mutant cell type.
4. Make plating bacteria of 7136.
- a) Inoculate 100 mL LB with 1 mL 7136 ON, incubate with shaking at 37 °C, for about 2.5 hr. (until the cells are in log phase).
- b) Pellet cells at 10K, 4 °C for 10 min.
- c) Resuspend the cell pellet in 3 mL LB liquid media.
5. Make plating bacteria of 7155.
- a) Inoculate 100 mL LB with 1 mL 7155 ON, incubate with shaking at 37 °C, for about 2.5 hr. (until the cells are in log phase).
- b) Pellet cells at 10K, 4 °C for 10 min.
- c) Resuspend the cell pellet in 3 mL LB liquid media.
6. Titer P22 phage on 7136 and on 7155.
7. Prepare P22 stock
- a) Inoculate 30 mL LB with 0.5 mL fresh 7155 ON.
- b) Infect with 1 plaque from Day 3 titer.
- c) Shake at 37 °C for 4 hr. (Turbid cell growth should turn clear, as a result of cell lysis).
- d) Transfer culture to centrifuge tube and spin at 10K, 4 °C for 10 min to pellet cell debris.
- e) Decant supernatant and spin it at 15K, 4 °C for 45 min to pellet phage.
- f) Resuspend phage pellet in NMT buffer at 4 °C ON.
8. Titer phage stock on 7136 and 7155.
9. Inoculate 30 mL LB with one loopful of 7136 ON and shake at 37 °C ON.
10. Make 1 L LB super broth in a 4 L flask.
11. Large scale preparation of P22 phage
- a) Inoculate 1 L superbroth with 10 mL of fresh 7136 ON.
- b) Incubate with shaking at 37 °C.
- c) Using a microscope, periodically check cells for appearance, activity and number. (They should be roundish and very active. It is good to check every 20 min or so.)
- d) When the O.D. = 2 (4 x 108 cells/mL), infect with the appropriate volume of phage stock to get an M.O.I. = 3-5. (320 cells/large square = 20 cells/small square = 4 x 108 cells/mL)
- e) Continue to shake at 37 °C and check cells under microscope for about 3 hr. Infected cells should become sluggish and stuck together in chains. The odor of infected cells is quite distinct also.
- f) Transfer flask of cells to ice for about 20-30 min.
- g) Spin infected cell culture at 10K, 4 °C for 10 min.
- h) Decant super and discard. Put cells on ice.
- i) Resuspend cells in NMT buffer (about 10 mL per centrifuge bottle, 40 mL total). Vigorous shaking will be necessary to resuspend the cells.
- j) Transfer the cell suspension into a 50 mL tube and freeze in liquid N2. If the protocol cannot be finished right away, this is a good place to stop until the protocol can be completed.
- k) Add 1 mL CHCl3 and 100 mL of 20 mg/mL PMFS in DMSO.
- l) Thaw cells by shaking at 37 °C.
- m) Freeze again in liquid N2 and thaw again by shaking at 37 °C. Suspension should be viscous.
- n) Pellet cell debris by spinning at 10 K, 4 °C for 15-45 min.
- o) Decant and save super.
- p) Spin phage-containing super at 35K, 4 °C for 45 min.
- q) Resuspend phage pellet in small volume (1-5 mL) of NMT buffer, at 4 °C, ON.
12. Spin phage solution at 7K, 4 °C for 10 min to pellet excess debris.
13. Add RNAse to a final concentration of 0.2 mg/mL. Incubate at 37 °C for 20-30 min.
14. Run the phage solution on a CsCl step gradient. Use NMT buffer to make CsCl solutions! The Mg2+ ion concentration must be maintained in order to keep the DNA packaged inside the phage capsid.
- a) Prepare 10 mL of 3 CsCl solutions. r = 1.3, r = 1.5 and r = 1.7.
- b) Prepare 6 mL of CsCl solution r = 1.4, by mixing equal volumes of the appropriate CsCl solutions.
- c) Prepare 6 mL of CsCl solution r = 1.6, by mixing equal volumes of the appropriate CsCl solutions.
- d) In a centrifuge tube, layer appropriate volumes of the 5 CsCl solutions and top off with the phage solution. The total volume of CsCl and phage solutions should be equal to the volume of the tube.
- e) Spin at 28 K for 4.5 hr, using a slow acceleration (set at 4) and no brake (set at 0).
15. Collect the phage band. The phage solution has a density of about 1.5. The band should be bluish-white.
16. Dialyze the phage-CsCl solution against NMT buffer ON at 4 °C.
17. Continue dialysis. Change buffer twice on this day.
18. Change dialyzing buffer and continue dialysis for a couple of hours.
19. Collect phage solution from dialysis tubing. Spin at 28 K for 20 min. May need to spin longer to pellet phage.
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