Griffitts:Transduction

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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)

LB Na-citrate (75 mL)

LB-MC Top Agar (250 mL)

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

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