Sauer:P1vir phage transduction

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Lysate preparation

1. Dilute an overnight culture (LB medium) of donor strain 1:100 in fresh LB + 5 mM CaCl2 and 0.2% glucose (2.5 mL should be enough). Grow with aeration at 37 ˚C for 1 hr. Add 100 µL of P1 phage lysate to the culture, continue growing at 37 ˚C. Monitor for 1–3 hr until the culture has lysed completely.

2. Add several drops of chloroform to the lysate and vortex. Centrifuge away the debris (14,000 rpm, 1–2 min) and transfer the supernatent to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.

Transduction

1. Grow recipient strain overnight in LB medium (2 mL culture is plenty).

2. On the next day, harvest the cells by centrifugation (6000 rpm, 2 min) and resuspend in original culture volume in fresh LB + 100 mM MgSO4 + 5 mM CaCl2. (note: 10 mM MgSO4 works fine, too, so you can use the 0.1 M MgSO4 the kitchen makes.)

3. Set up four "reactions":

A. 100 µL undiluted P1 lysate + 100 µL recipient cells B. 100 µL 1:10 diluted P1 lysate + 100 µL recipient cells C. 100 µL LB + 100 µL recipient cells D. 100 µL undiluted P1 lysate + 100 µL LB

(note for step 3: LB = LB + 100 mM MgSO4 + 5 mM CaCl2; dilute your P1 lysate in this as well)

4. Incubate tubes at 37 ˚C for 30 min.

5. Add 200 µL Na-Citrate (pH 5.5), then add 1 mL LB (the real thing this time) and incubate at 37 ˚C for 1 hr to allow expression of the antibiotic resistance marker.

6. Spin cells at 6000 rpm for 2-3 min.

7. Resuspend each in 100 µL LB + 100 mM Na-Citrate (pH 5.5) and plate all of it on an appropriate antibiotic-containing plate.

Who to ask about this protocol

Chris, Sean, Kathleen


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