Difference between revisions of "M13 Bacteriophage Production Protocol"
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| − | This protocol describes how to produce plaques using M13 bacteriophage, and how to then harvest phage from these plaques. This is a multistep process which requires making both bottom agar (a lower layer of agar with antibiotics) and top agar (a top layer of agar with MgCl2). | + | This protocol describes how to produce plaques using M13 bacteriophage, and how to then harvest phage from these plaques. This is a multistep process which requires making both bottom agar (a lower layer of agar with antibiotics) and top agar (a top layer of agar with MgCl2), as well as growing host cells and performing phage dilutions. |
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== Preparing Phage == | == Preparing Phage == | ||
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1. Grow 3mL of cells with phage plasmid in liquid media overnight. These cells produce M13 phage which can then be isolated. For example, grow J100265 + J100270 S1030 cells in 50ug/mLCarb + 50ug/mL Kan + 10ug/mL Tet for SPT7-spec phage. | 1. Grow 3mL of cells with phage plasmid in liquid media overnight. These cells produce M13 phage which can then be isolated. For example, grow J100265 + J100270 S1030 cells in 50ug/mLCarb + 50ug/mL Kan + 10ug/mL Tet for SPT7-spec phage. | ||
| − | 2. Centrifuge 1.5mL of cells at full speed (~15,500 RPM) for at least two minutes. The cells will be spun down into a pellet, while the phage will remain in the supernatant. Save the supernatant in a separate microfuge tube. The phage will remain viable for weeks if stored at 4C. | + | 2. Centrifuge 1.5mL of cells at full speed (~15,500 RPM) for at least two minutes. The cells will be spun down into a pellet, while the phage will remain in the supernatant. Save the supernatant in a separate microfuge tube and discard the tube with the pelleted cells. The phage will remain viable for weeks if stored at 4C. |
| + | 3. When preparing to plate phage, make serial dilutions of the phage stock in LB broth. Each dilution should have a final volume of >10uL. Create a broad range of dilutions for plating, as too high of a phage concentration will produce many small, crowded plaques, while too low of a dilution may not produce plaques. Generally, a range from 10^-3 to 10^-9 should be sufficient. | ||
== Preparing Top and Bottom Agar == | == Preparing Top and Bottom Agar == | ||
Revision as of 22:07, 17 May 2017
This protocol describes how to produce plaques using M13 bacteriophage, and how to then harvest phage from these plaques. This is a multistep process which requires making both bottom agar (a lower layer of agar with antibiotics) and top agar (a top layer of agar with MgCl2), as well as growing host cells and performing phage dilutions.
Preparing Phage
1. Grow 3mL of cells with phage plasmid in liquid media overnight. These cells produce M13 phage which can then be isolated. For example, grow J100265 + J100270 S1030 cells in 50ug/mLCarb + 50ug/mL Kan + 10ug/mL Tet for SPT7-spec phage.
2. Centrifuge 1.5mL of cells at full speed (~15,500 RPM) for at least two minutes. The cells will be spun down into a pellet, while the phage will remain in the supernatant. Save the supernatant in a separate microfuge tube and discard the tube with the pelleted cells. The phage will remain viable for weeks if stored at 4C.
3. When preparing to plate phage, make serial dilutions of the phage stock in LB broth. Each dilution should have a final volume of >10uL. Create a broad range of dilutions for plating, as too high of a phage concentration will produce many small, crowded plaques, while too low of a dilution may not produce plaques. Generally, a range from 10^-3 to 10^-9 should be sufficient.
