20.109(F07): Growth of phage materials

Protocols

In advance of this lab, a bacterial host (namely XL1-blue) was infected with the modified M13 phage called "3-12." Though the precise details of the 3-12 modification are still unpublished, it was isolated from a screen for changes in the M13 p8 that enable the phage to bind a metal (Iridium). Today you will harvest the phage from the supernatant of the infected bacterial culture and then titer it.

Part 1: Phage purification

1. Spin 2 eppendorf tubes with infected cells in a room temperature microfuge, 1 minute at full speed.
2. Remove the supernatant to clean fresh eppendorf tubes.
3. Use your P1000 to measure the volume, then add a 1/6th volume of 20% PEG-8000/2.5M NaCl solution.
4. Invert to mix then incubate on ice 60 minutes.
5. Spin in a room temperature microfuge, 15 minutes at full speed. A white pellet should be visible...these are your precipitated phage.
6. Remove the supernatant by aspiration (carefully so as not to disturb the pellet) or using your P1000.
7. Spin the tubes 1 minute more to pellet any droplets stuck to the walls of the tube and use your P200 to remove the last drops of liquid from the pellet.
8. Resuspend the pellet in 100 ul sterile H2O, pool the volumes of both eppendorf tubes into one and add a 1/6th volume of 20% PEG-8000/2.5M NaCl solution.
9. Invert to mix then incubate on ice, 15 minutes.
10. Balance your sample against that of another group, or against an eppendorf with water. Spin in a room temperature microfuge, 10 minutes at full speed.
11. Aspirate the supernatant and resuspend the pellet in 100 ul sterile H2O.
12. If the solution looks at all cloudy, spin in a room temperature 1 minute more and move supernatant with the phage to a new eppendorf tube.

Part 3: Titering phage

This protocol is identical to one you performed earlier in the term. You should dilute the phage you've prepared today so the final concentrations of phage are 10^6, 10^8, and 10^10th less concentrated than your stock. In addition to 10 ul of these three dilutions, you should include a "no phage" sample.

Before you leave you should place your petri dishes in the 37° incubator and give the phage stock to the teaching faculty, who will store it at 4° for you until next time.

DONE!