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)
- 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: