Difference between revisions of "M13 Bacteriophage Production Protocol"
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1. Bottom agar should be produced following the same recipe for creating plates for plating cells. Plates with the appropriate antibiotics that were already made for other purposes are acceptable. There’s no need to pour plates with a thinner layer of agar. Bottom agar should have the antibiotics that the host cells are resistant to, but not the antibiotics that the phage genome creates resistance for. For example, if using J100265 cells, bottom agar should have 50ug/mL carbenicillin, but not kanamycin. If host cells are S1030s, bottom agar should also have 10ug/mL tetracycline to select for the F’ plasmid. | 1. Bottom agar should be produced following the same recipe for creating plates for plating cells. Plates with the appropriate antibiotics that were already made for other purposes are acceptable. There’s no need to pour plates with a thinner layer of agar. Bottom agar should have the antibiotics that the host cells are resistant to, but not the antibiotics that the phage genome creates resistance for. For example, if using J100265 cells, bottom agar should have 50ug/mL carbenicillin, but not kanamycin. If host cells are S1030s, bottom agar should also have 10ug/mL tetracycline to select for the F’ plasmid. | ||
| − | 2. Make top agar with 7g/L LB agar, 20g/L LB broth, and 1M MgCl2. Autoclave top agar, then transfer in 3mL aliquots to sterile tubes. Incubate in a water bath at 47°C until use. | + | 2. Make top agar with 7g/L LB agar, 20g/L LB broth, and 1M MgCl2. Autoclave top agar, then transfer in 3mL aliquots to sterile tubes. Incubate in a water bath at 47°C until use. Make certain that the top agar has cooled to 47°C if it’s being used immediately after autoclaving. If top agar is too hot, it will kill host cells and phage. |
== Plating Top Agar with Phage and Host Cells == | == Plating Top Agar with Phage and Host Cells == | ||
Revision as of 22:16, 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 Kanamycin to produce 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 4°C.
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
1. Bottom agar should be produced following the same recipe for creating plates for plating cells. Plates with the appropriate antibiotics that were already made for other purposes are acceptable. There’s no need to pour plates with a thinner layer of agar. Bottom agar should have the antibiotics that the host cells are resistant to, but not the antibiotics that the phage genome creates resistance for. For example, if using J100265 cells, bottom agar should have 50ug/mL carbenicillin, but not kanamycin. If host cells are S1030s, bottom agar should also have 10ug/mL tetracycline to select for the F’ plasmid.
2. Make top agar with 7g/L LB agar, 20g/L LB broth, and 1M MgCl2. Autoclave top agar, then transfer in 3mL aliquots to sterile tubes. Incubate in a water bath at 47°C until use. Make certain that the top agar has cooled to 47°C if it’s being used immediately after autoclaving. If top agar is too hot, it will kill host cells and phage.
