Griffitts:Transduction

ΦN3 and ΦM12 Phage Transduction (S. meliloti)

Preparation of lysate

• Set up 24-hr LB-MC culture of each donor strain, so it reaches OD₆₀₀ = ~1.0
• Dilute phage stock appropriately (10^-4 if standard lysate) in LB-MC
• Mix 20 μL of the phage dilution with 400 μL bacterial culture in a 15-mL tube
• Incubate at 30°C for 30 min
• Add 10 mL of 42°C LB-MC Top agar
• Invert three times
• Dump contents into an empty Petri plate
• Allow to solidify
• Incubate at 30°C overnight

Phage Harvesting

• Plaques should be confluent
• Add 2 mL LB-MC to the plate
• Mash the top agar for 2 min with spreader
• Recover 1.5 mL to microfuge tube
• Spin down debris
• Move 800 μL of phage-containing supernatant to a new microfuge tube
• Add 50 μL CHCl₃ (chloroform)
• Vortex
• Briefly centrifuge
• Store at 4°C
• This stock is about 10¹⁰ PFU/mL

Transduction

• Grow each recipient strain to OD₆₀₀ = 1.0 (10⁹ CFU/mL) in LB-MC
• Combine 30 μL or 90 μL of lysate with 1 mL of culture (one may work better)
• Incubate at 30°C for 30 min
• Centrifuge and resuspend in LB (no calcium) 3 times
  • This washes away calcium and halts phage adsorption
• Centrifuge and resuspend in 70 μL of LB
• Plate on selective medium, LB (no calcium)

ΦP1vir Phage Transduction (E. coli)

Preparation of lysate

• Set up a 24-hr LB-MC culture of each donor strain, so it reaches OD₆₀₀ = ~1.0–2.0
• Dilute Phage stock appropriately (10^-2 or 10^-3 if standard lysate) in LB-MC
• Mix 50 μL of the phage dilution with 500 μL bacterial culture in a 15-mL tube
• Incubate at 37°C for 25 min
• Add 10 mL of 42°C LB-MC Top Agar
• Invert twice
• Dump contents into an empty Petri plate
• Allow to solidify
• Incubate at 37°C overnight

Phage Harvesting

• Plaques should be confluent (be aware they are tiny)
• Add 3 mL LB-MC
• Mash the top agar for 5 min with spreader, until it is a smooth homogenate
• Recover 1 mL to a new microfuge tube
• Centrifuge for 5 min at 13,200 rpm
• Move phage-containing supernatant (~1 mL) to a new microfuge tube
• Add 50 μL CHCl₃ (chloroform)
• Vortex
• Briefly centrifuge
• Store at 4°C
• This stock is about 10⁹ PFU/mL (?)

Transduction

• Set up a 24-hr LB-MC culture of each recipient strain, so it reaches OD₆₀₀ = ~1.0–2.0
• Combine 20 μL or 100 μL of undiluted lysate with 1 mL of culture (one may work better)
• Incubate on rack at 37°C for 25 min*
• Centrifuge
• Resuspend cells in 800 μL of LB-citrate
• Shake at 37°C for 60 min*
• Centrifuge
• Resuspend in 150 μL of LB-citrate
• Plate entire sample on selective medium (e.g. LB-Km (no calcium)

• NOTE: The recovery period is important at least for Km markers

Solutions

LB MC Broth (75 mL)

• 75 mL sterile LB
• 0.3 mL 2 M MgSO₄
• 0.3 mL 1 M CaCl₂

LB Na-citrate (75 mL)

• 75 mL sterile LB
• 2 mL 1 M Na-citrate

LB-MC Top Agar (250 mL)

• 250 mL LB Top Agar

Boil to liquify
Place in 42°C water bath to cool
When cool, add:

• 1 mL 2 M MgSO₄
• 1 mL 1 M CaCl₂