IGEM:Paris Bettencourt 2012/Protocols/P1 trans

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Protocol: P1 Transduction

This protocol uses a phage to transfer marker from a donor strain to a recipient strain. The phage head packages about 90 kb of DNA, so donor DNA near the marker is also transferred. Note that this can cause problems if you are working with several markers that are very close together.

Lysate preparation

Note: P1 phage should be stored at 4°C. It can't be frozen.

  1. The night before, start a 5 mL culture of the donor strain in selective LB.
  2. The day of, dilute the donor strain 1:100 into Phage Lysis medium
  • 50 uL of cells in 5 mL LB
  • 50 uL of 20% glucose
  • 50 uL of 1M MgSO4
  • 25 uL of 1M CaCl2
  • No antibiotics!!
  1. Incubate at 37°C for 1 hour
  2. Add 50 uL of P1 phage lysate. Monitor the culture for 1-3 hours. The culture should become cloudy, then clear following lysis.
  3. Add 500 uL of chloroform to the lysate and vortex.
  4. Centrifuge at max speed for 1 minute to clear the cell debris. Collect the supernatant.
  5. Phage lysate can be stored indefinitely at 4°C. Freezing will destroy the phage.

Transduction

  1. The night before, start a 5 mL culture of the recipient strain in selective LB.
  2. The day of, harvest the cells by centrifugation (6000 rpm for 2 min).
  3. Resuspend in original culture volume in 5 mL Phage Infection LB.
  • 5 mL LB
  • 50 uL of 1M MgSO4
  • 25 uL of 1M CaCl2
  1. Transfer 100 uL of donor P1 lysate per transformation to a 1.5 mL tube.
  2. Set up 4 reactions for each transduction.
    1. 100 uL donor lysate + 100 uL recipient cells
    2. 10 uL donor lysate + 190 uL recipient cells
    3. 100 uL donor lysate + 100 uL plain LB
    4. 100 uL plain LB + 100 uL recipient cells
  3. Incubate at 37°C for 30 minutes.
  4. Stop the infection with 200 uL of 1M sodium citrate (pH 5.5).
  5. Add 1 mL LB and recover the cells for 1-2 hours.
  6. Spin the cells down and resuspend for plating. 100 uL LB + 10 uL of 1M sodium citrate (pH 5.5).
  7. Plate on selective LB.
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