Phage Lysate Preparation

Materials

• Overnight culture of a permissive E. coli host (see the table below)
• Frozen stock of phage (or plate with fresh plaques)
• LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)
• 50 mL flasks (recommended) or test tubes
• 15 mL Falcon tubes
• Shaking incubator at appropriate temperature (37° or 30°)
• Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)

Procedure

T7 lysate showing small amount of chloroform that has settled to the bottom of the tube.

1. Prepare overnight culture of permissive host
2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
3. Grow these cultures for 30-60 min (OD ~0.2)
   • It's highly recommended that you start a no-phage control at the same time (i.e., an extra flask of diluted host w/no phage added) for turbidity comparison w/ lysed cultures
4. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~10⁶ PFU.
5. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
   • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
6. Add 50-100 µL of chloroform and vortex
7. Spin 5min @ 5000 × g (RCF)
8. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50 µL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
9. Determine phage titer Protocol: Measuring Phage Titers

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

• T4 lysates (10¹⁰-10¹² PFU/ml)
• T5 and T6 lysates (10⁷-10⁹ PFU/ml)
• T7 lysates (???? PFU/ml)

	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) immediately after phage is added from a high-titer lysate.
	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) 3 hours later showing that lysis has occurred in the flask with phage but cell density has increased in the negative control with only E. coli

Variants

nsAA evolution strains

• Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
• Supplement media with antibiotics and 250μM nsAA.
• Cultures of this strain may need up to 24h to reach saturation.
• Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

Phage Lysate Preparation

Materials

• Overnight culture of a permissive E. coli host (see the table below)
• Frozen stock of phage (or plate with fresh plaques)
• LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)
• 50 mL flasks (recommended) or test tubes
• 15 mL Falcon tubes
• Shaking incubator at appropriate temperature (37° or 30°)
• Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)

Procedure

T7 lysate showing small amount of chloroform that has settled to the bottom of the tube.

1. Prepare overnight culture of permissive host
2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
3. Grow these cultures for 30-60 min (OD ~0.2)
   • It's highly recommended that you start a no-phage control at the same time (i.e., an extra flask of diluted host w/no phage added) for turbidity comparison w/ lysed cultures
4. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~10⁶ PFU.
5. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
   • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
6. Add 50-100 µL of chloroform and vortex
7. Spin 5min @ 5000 × g (RCF)
8. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50 µL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
9. Determine phage titer Protocol: Measuring Phage Titers

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

• T4 lysates (10¹⁰-10¹² PFU/ml)
• T5 and T6 lysates (10⁷-10⁹ PFU/ml)
• T7 lysates (???? PFU/ml)

	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) immediately after phage is added from a high-titer lysate.
	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) 3 hours later showing that lysis has occurred in the flask with phage but cell density has increased in the negative control with only E. coli

Variants

nsAA evolution strains

• Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
• Supplement media with antibiotics and 250μM nsAA.
• Cultures of this strain may need up to 24h to reach saturation.
• Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

Phage Lysate Preparation

Materials

• Overnight culture of a permissive E. coli host (see the table below)
• Frozen stock of phage (or plate with fresh plaques)
• LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)

• 15 mL Falcon tubes
• Shaking incubator at appropriate temperature (37° or 30°)
• Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)

Procedure

T7 lysate showing small amount of chloroform that has settled to the bottom of the tube.

1. Prepare overnight culture of permissive host
2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
3. Grow these cultures for 30-60 min (OD ~0.2)
   • It's highly recommended that you start a no-phage control at the same time (i.e., an extra flask of diluted host w/no phage added) for turbidity comparison w/ lysed cultures
4. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~10⁶ PFU.
5. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
   • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
6. Add 50-100 µL of chloroform and vortex
7. Spin 5min @ 5000 × g (RCF)
8. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50 µL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
9. Determine phage titer Protocol: Measuring Phage Titers

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

• T4 lysates (10¹⁰-10¹² PFU/ml)
• T5 and T6 lysates (10⁷-10⁹ PFU/ml)
• T7 lysates (???? PFU/ml)

	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) immediately after phage is added from a high-titer lysate.
	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) 3 hours later showing that lysis has occurred in the flask with phage but cell density has increased in the negative control with only E. coli

Variants

nsAA evolution strains

• Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
• Supplement media with antibiotics and 250μM nsAA.
• Cultures of this strain may need up to 24h to reach saturation.
• Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

Phage Lysate Preparation

Materials

• Overnight culture of a permissive E. coli host (see the table below)
• Frozen stock of phage (or plate with fresh plaques)
• LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)
• 50 mL flasks (recommended) or test tubes * 15 mL Falcon tubes
• Shaking incubator at appropriate temperature (37° or 30°)
• Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)

T7 lysate showing small amount of chloroform that has settled to the bottom of the tube.

1. Prepare overnight culture of permissive host
2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
3. Grow these cultures for 30-60 min (OD ~0.2)
   • It's highly recommended that you start a no-phage control at the same time (i.e., an extra flask of diluted host w/no phage added) for turbidity comparison w/ lysed cultures
4. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~10⁶ PFU.
5. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
   • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
6. Add 50-100 µL of chloroform and vortex
7. Spin 5min @ 5000 × g (RCF)
8. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50 µL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
9. Determine phage titer Protocol: Measuring Phage Titers

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

• T4 lysates (10¹⁰-10¹² PFU/ml)
• T5 and T6 lysates (10⁷-10⁹ PFU/ml)
• T7 lysates (???? PFU/ml)

	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) immediately after phage is added from a high-titer lysate.
	Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) 3 hours later showing that lysis has occurred in the flask with phage but cell density has increased in the negative control with only E. coli

Variants

nsAA evolution strains

• Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
• Supplement media with antibiotics and 250μM nsAA.
• Cultures of this strain may need up to 24h to reach saturation.
• Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

Phage Lysate Preparation

Materials

• Overnight culture of a permissive E. coli host (see the table below)
• Frozen stock of phage (or plate with fresh plaques)
• LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)

• Shaking incubator at appropriate temperature (37° or 30°)
• Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)

Procedure:

1. Prepare overnight culture of permissive host
2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
3. Grow these cultures for 30-60 min (OD ~0.2)
   • It's highly recommended that you start a no-phage control at the same time (i.e., an extra flask of diluted host w/no phage added) for turbidity comparison w/ lysed cultures
4. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~10⁶ PFU.
5. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
   • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
6. Add 50-100 µL of chloroform and vortex

1. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50 µL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
2. Determine phage titer Protocol: Measuring Phage Titers

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

• T4 lysates (10¹⁰-10¹² PFU/ml)
• T5 and T6 lysates (10⁷-10⁹ PFU/ml)
• T7 lysates (???? PFU/ml)

Variants

nsAA evolution strains

• Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
• Supplement media with antibiotics and 250μM nsAA.
• Cultures of this strain may need up to 24h to reach saturation.
• Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

Phage Lysate Preparation

Materials

• Overnight culture of a permissive E. coli host (see the table below)
• Frozen stock of phage (or plate with fresh plaques)
• LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)

• Shaking incubator at appropriate temperature (37° or 30°)
• Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)

Procedure:

1. Prepare overnight culture of permissive host
2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
3. Grow these cultures for 30-60 min (OD ~0.2)

1. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~10⁶ PFU.
2. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
   • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
3. Add 50-100 µL of chloroform and vortex
4. Spin 5min @ 5000 &multiply; g (RCF)
5. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50 µL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
6. Determine phage titer Protocol: Measuring Phage Titers

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

• T4 lysates (10¹⁰-10¹² PFU/ml)
• T5 and T6 lysates (10⁷-10⁹ PFU/ml)
• T7 lysates (???? PFU/ml)

Variants

nsAA evolution strains

• Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
• Supplement media with antibiotics and 250μM nsAA.
• Cultures of this strain may need up to 24h to reach saturation.
• Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

Phage Lysate Preparation

Procedure:

• • Can be useful to start an no-phage control (i.e. diluted host w/ no phage) for turbidity comparison w/ lysed cultures

1. Transfer lysate (supernatant) to a clean tube

• • For long-term storage @ -80°C, add glycerol to 20% and mix well

1. Determine phage titer Protocol: Measuring Phage Titers

Phage Lysate Preparation

• Definitely works with T4 and T7, should work with most E. coli phages (although lysis times and final titers may differ)

Reagents / Materials:

• Overnight culture of a permissive host
• BL21 usually works well for T7 and T7Δ2 (a.k.a mutator); MJH116(Amberless) with appropriate nsAA-aaRS for nsAA evolution strains
• Frozen Stock of Phage (or fresh plating)
• LB Media (w/ antibiotics or supplements appropriate for host)
• 50mL flasks or test tubes
• Shaking incubator at appropriate temp (37° or 30°) *Chloroform (optional but recommended)

Procedure:

1. Prepare overnight culture of permissive host (Amberless aaRS strains should be grown w/ 250μM nsAA in media and may need up to 24h to reach saturation)
2. Dilute 1mL of overnight culture into 10mL fresh media (1:10 dilution), grow for 30-60min (OD ~0.2)
   • Some finicky strains (e.g. nsAA evolved strains) apparently lyse stochastically, so best to set up 8-10 lysis cultures (lyses?)
   • Can be useful to start an no-phage control (i.e. diluted host w/ no phage) for turbidity comparison w/ lysed cultures
3. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip.
4. Shake @~200RPM at the appropriate temperature until culture lyses completely
   • i.e. little or no turbidity compared to no-phage control; will usually see bits of aggregated cell debris also
5. Add 50-100uL of chloroform and vortex
6. Spin 5min @ 5000RPM
7. Transfer lysate (supernatant) to a clean tube
   • For long-term storage @ 4°C, add 50μL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
8. Determine phage titer Protocol: Measuring Phage Titers

-- Main.ColinBrown - 14 Dec 2017