20.109(F07): Growth of phage materials: Difference between revisions
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==Introduction== | ==Introduction== | ||
==Protocols== | ==Protocols== | ||
In advance of this lab, a bacterial host (namely [ | In advance of this lab, a bacterial host (namely [[E. coli genotypes| 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 Though the precise details of the 3-12 modification are still unpublished, it was isolated from a screen for changes in the M13 p8 that enable the phage to bind a metal (Iridium).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=== | ===Part 1: Phage purification=== | ||
#Spin 2 eppendorf tubes with infected cells in a room temperature microfuge, 1 minute at full speed. | #Spin 2 eppendorf tubes with infected cells in a room temperature microfuge, 1 minute at full speed. | ||
| Line 16: | Line 16: | ||
#Aspirate the supernatant and resuspend the pellet in 100 ul sterile H2O. | #Aspirate the supernatant and resuspend the pellet in 100 ul sterile H2O. | ||
#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. | #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 2: Titering phage=== | ===Part 2: Titering phage=== | ||
This protocol is identical to one you performed [[20.109(F07): Agarose gel electrophoresis| 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. | |||
DONE! | DONE! | ||
==For next time== | ==For next time== | ||
==Reagents list== | ==Reagents list== | ||
Revision as of 08:15, 12 July 2007
Introduction
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 Though the precise details of the 3-12 modification are still unpublished, it was isolated from a screen for changes in the M13 p8 that enable the phage to bind a metal (Iridium).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
- Spin 2 eppendorf tubes with infected cells in a room temperature microfuge, 1 minute at full speed.
- Remove the supernatant to clean fresh eppendorf tubes.
- Use your P1000 to measure the volume, then add a 1/6th volume of 20% PEG-8000/2.5M NaCl solution.
- Invert to mix then incubate on ice 60 minutes.
- Spin in a room temperature microfuge, 15 minutes at full speed. A white pellet should be visible...these are your precipitated phage.
- Remove the supernatant by aspiration (carefully so as not to disturb the pellet) or using your P1000.
- 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.
- 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.
- Invert to mix then incubate on ice, 15 minutes.
- 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.
- Aspirate the supernatant and resuspend the pellet in 100 ul sterile H2O.
- 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 2: 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.
DONE!
