Generating High-Titer-High Volume Transduction Phage Lysates from GNF phage lysates (reamplification)
08/22/13 Linda Berney-Meyer, adapted from Zaritza Petrova’s protocol (Hatfull Lab)
Materials:
- mc2155 cells (OD~1.0)
- mc2155 medium
- MP Buffer
- Topagar
- 7H10 plates (plus glycerol) plates (15cm and regular for titration)
- 10cm syringe
- 0.22μm filter
Protocol:
Infection
Note: It is even better to use cells that have been subcultured in 7H9 medium without tween. Although the cells will be clumpy. The infection is better since tween inhibits the phage receptors and thereby adsoprtion
- Use 5/10 μl to infect 300μl mc 2155 cells OD~1.0 which have been washed with MP buffer. Let infect 30min at room temperature.
Note: It is even better to use cells that have been subcultured in 7H9 medium without tween. Although the cells will be clumpy. The infection is better since tween inhibits the phage receptors and thereby adsoprtion
- Melt top agar. Add ~8ml-10ml top agar to a capped tube. Add the cell+phage mix to tube and spread immediately onto a 15cm petri dish. Place at 30°C for 2-3days.
Harvest
- Inspect the lawn: the best lawns are when the plate is lacy. However, a completely clear plate after 3 days can give titers of 1011pfu/ml as well. I harvest all plates with plaques >100, because a low titer can be used for sequential titer boosts.
- Add 12ml MP buffer. Let plates shake at 80rpm for ~2h to help the buffer spread nicely throughout the plate. Collect with 10cm syringe and filter through 0.22μm filter.
- Take out 10μl lysate into 90μl buffer. Make serial dilutions (I go up to 10 -8). Spot 2μl of each dilution onto mc 2155 lawn -Note: I usually prepare a titering 96-well plate or you can also spot onto round plates.
- Incubate plate at 30°C for 2 days or until plaques are countable. - Note: To check for lysate purity and revertants, I spot from the same dilutions on a second plate and incubate at 37° as well.
- Determine titer
Titer Boost
- Some of the titers first achieved are 109 pfu/ml or less. For those, a titer boost can give the desired 1010 pfu/ml or higher. Usually, a boost can generate a titer 2-3 logs higher than the initial one
- Infection and plating is the same as described above (30min time to infect at room temperature, followed by plating on a large petri dish and incubating at 30°C 2-3 days) with the following volumes.
