Griffitts:Transduction

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)

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

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)
Boil to liquify Place in 42°C water bath to cool When cool, add:
 * 250 mL LB Top Agar
 * 1 mL 2 M MgSO4
 * 1 mL 1 M CaCl2