Difference between revisions of "20.109(F07): Growth of phage materials"
From Course Wiki
Line 2: | Line 2: | ||
==Introduction== | ==Introduction== | ||
==Protocols== | ==Protocols== | ||
− | In advance of this lab, a bacterial host (namely [http:// | + | In advance of this lab, a bacterial host (namely [http://openwetware.org/wiki/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 Though the precise details of the 3-12 modification are still unpublished, it was isolated from a screen for changes in the M13 p8 that enable the phage to bind a metal (Iridium).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. | ||
#Remove the supernatant to clean fresh eppendorf tubes. | #Remove the supernatant to clean fresh eppendorf tubes. | ||
− | #Use your P1000 to measure the volume, then add a 1/6th volume of | + | #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. | #Invert to mix then incubate on ice 60 minutes. | ||
− | #Spin in a room temperature microfuge, 15 minutes at full speed. | + | #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. | ||
+ | |||
Revision as of 15:07, 12 July 2007
Contents
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
DONE!