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). | ||
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== Preparing Phage == | == Preparing Phage == | ||
| − | 1. Grow 3mL of cells with phage plasmid in liquid media overnight. These cells | + | 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 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. The phage will remain viable for weeks if stored at 4C. |
== Preparing Top and Bottom Agar == | == Preparing Top and Bottom Agar == | ||
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== Plating Top Agar with Phage and Host Cells == | == Plating Top Agar with Phage and Host Cells == | ||
Revision as of 22:03, 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).
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. The phage will remain viable for weeks if stored at 4C.
