Genetics - Transduction with Bacteriophage P22

Jeff Lawrence (G03)

1. Grow recipient cells overnight in appropriate medium.

2. Dilute donor bacteriophage to 108 pfu/mL, about a 10-2 dilution.

3. Mix 100 µL cells (108 cells) and 100 µL of the phage dilution (107 phage). This allows a multiplicity of infection of 0.1 phage per cell. For two-fragment transduction, increase MOI to between 1 and 5.

4. Incubate at 37°C for 30 min (Tc, Kn, Rf, Gn) or 60 min (Cm) with shaking.

5. Plate directly on selective medium. Adding EGTA to 10 mM final concentration in plates will decrease phage infection on the plates.

6. Incubate overnight at 37°C. The protocol will yield between 100 and 500 transductants.

7. Pick transductants early to avoid excessive phage contamination. Use a toothpick to select cells from the top of a colony, avoiding the surrounding media. Streak for single colonies on green indicator plates. Streak at least 4 transductants; more are needed if weakly linked, unselected markers are required. Grow plates overnight at 37 °C.

8. Dilute P22 strain H5 ten-fold in T2 buffer. Prepare a green plate with ~20 µL H5 struck in a line down the center of the plate.

9. Select a large, light colony from each green plate streak with a toothpick. Small, dark colonies are infected with phage. Mark two master plates with the toothpick then streak across the H5 bacteriophage on the green plate. Multiple cross streak may be placed on a single green plate. Incubate overnight.

10. Print one master to appropriate media to verify phenotypes of transductants. After printing, select a strain from the second master that was infected upon cross-streaking (produced small, dark patches following the H5 cross-streak). Grow strain and freeze.

Last Update: Thursday, 19-Jun-2014 11:54:43 PDT
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