M13 Bacteriophage Production Protocol - Revision history

Macampbell at 15:54, 7 June 2017

2017-06-07T15:54:17Z

2017-06-07T15:53:47Z

2017-05-17T23:01:10Z

2017-05-17T22:59:32Z

2017-05-17T22:58:27Z

2017-05-17T22:58:06Z

2017-05-17T22:57:53Z

2017-05-17T22:57:17Z

2017-05-17T22:54:50Z

2017-05-17T22:54:24Z

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 . 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.
-. Make top agar with 7g/L LB agar, 20g/L LB broth, and 5 mM 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.
+. Make top agar with 7g/L LB agar, 20g/L LB broth, and 5 mM MgCl<sub>2</sub>. 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 ==

@@ Line 14: / Line 14: @@
 . 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.
-. 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.
+. Make top agar with 7g/L LB agar, 20g/L LB broth, and 5 mM 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 ==

← Older revision		Revision as of 23:01, 17 May 2017
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−	== Summary ~~and Troubleshooting~~ ==	+	== Summary ==

	1. Grow S1030 cells with J100265 and J100270 plasmids overnight to produce SPT7-spec phage. J100269 or J100271 could take the place of the J100270 plasmid for producing promiscuous and wild type phage, respectively.		1. Grow S1030 cells with J100265 and J100270 plasmids overnight to produce SPT7-spec phage. J100269 or J100271 could take the place of the J100270 plasmid for producing promiscuous and wild type phage, respectively.

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 == Preparing Phage ==
-. Grow 3mL of cells with the phage plasmid in liquid media overnight.  These cells produce M13 phage which can then be isolated from the liquid media. For example, grow J100265 + J100270 S1030 cells in 50ug/mL carbenicillin + 50ug/mL kanamycin to produce SPT7-spec phage.
+. Grow 3mL of cells with the phage plasmid in liquid media overnight.  These cells produce M13 phage which can then be isolated from the liquid media. For example, grow J100265 + J100270 S1030 cells in LB + 50ug/mL carbenicillin + 50ug/mL kanamycin to produce SPT7-spec phage.
 . 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.

← Older revision		Revision as of 22:58, 17 May 2017
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	3. Save the microfuge tubes at 4°C. The phage in the cores will become suspended in the liquid media.		3. Save the microfuge tubes at 4°C. The phage in the cores will become suspended in the liquid media.

−	[[File:plaques.jpg\|500px\|thumb\|left\|Example Plate with Plaques]] [[File:plaques2.png\|500px\|thumb\|~~right~~\|Example Plate with Plaques]]	+	[[File:plaques.jpg\|500px\|thumb\|left\|Example Plate with Plaques]] [[File:plaques2.png\|500px\|thumb\|center\|Example Plate with Plaques]]

← Older revision		Revision as of 22:58, 17 May 2017
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	[[File:plaques.jpg\|500px\|thumb\|left\|Example Plate with Plaques]] [[File:plaques2.png\|500px\|thumb\|right\|Example Plate with Plaques]]		[[File:plaques.jpg\|500px\|thumb\|left\|Example Plate with Plaques]] [[File:plaques2.png\|500px\|thumb\|right\|Example Plate with Plaques]]
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		+
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	== Summary and Troubleshooting ==		== Summary and Troubleshooting ==

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 == Coring Plaques ==
-. After the plates have incubated for 12-18hrs, remove them from the incubator and inspect them for plaques. Plaques are small (1mm diameter) translucent circles. They are best seen when held up to light.
+. After the plates have incubated for 12-18hrs, remove them from the incubator and inspect them for plaques. Plaques are small (~1-3mm diameter) translucent circles. They are best seen when held up to light.
 . Identify a plate which has plaques that are clearly defined and isolated from one another. Prepare multiple 1.5mL microfuge tubes with 1mL of LB broth. Using an inverted 200uL pipette tip, press down on the agar over a plaque to take out a core of agar. Transfer the core into a prepared microfuge tube. Repeat this step for each microfuge tube. Try to keep this process as sterile as possible- for example, use forceps to remove the pipette tip from the box without grabbing the top, which will come in contact with the core.
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 . Save the microfuge tubes at 4°C. The phage in the cores will become suspended in the liquid media.
-[[File:plaques.jpg|500px|thumb|left|Example Plate with Plaques]]
+[[File:plaques.jpg|500px|thumb|left|Example Plate with Plaques]] [[File:plaques2.png|500px|thumb|right|Example Plate with Plaques]]
 == Summary and Troubleshooting ==

← Older revision		Revision as of 22:57, 17 May 2017
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	[[File:plaques.jpg\|500px\|thumb\|left\|Example Plate with Plaques]]		[[File:plaques.jpg\|500px\|thumb\|left\|Example Plate with Plaques]]
		+	[[File:plaques2.png\|500px\|thumb\|left\|Example Plate with Plaques]]

	== Summary and Troubleshooting ==		== Summary and Troubleshooting ==