Sauer:P1vir phage transduction

From OpenWetWare

Revision as of 09:45, 31 October 2005 by Smoore (Talk | contribs)
Jump to: navigation, search
SAUER LAB

Home        Protocols        Lab Members        Materials        Equipment        Links        Internal       

Contents

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 1 M 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.


Anecdotes

  • The chloroform used to sterilize the phage lysates, well, sterilizes. If you have visible chloroform drops in the lysate stock, don't add this to your recipient cells directly because you can kill a decent number of bacteria. Instead, aliquot your phage into microfuge tubes and incubate with the caps open at 37 ˚C for about 30 minutes to allow the chloroform to evaporate. Then add the recipient cells to the tubes with the phage.
  • When preparing the donor phage lysate, there is a huge variability in the titer of phage obtained at this step which makes transduction performance unpredictable. Some donor cells are slow "wake up" from stationary phase and 3 hours will not be enough. If it is obvious that there was no culture development in the tube, let it shake overnight. The next morning, you will have a culture of cells and, perhaps, noticeable cell debris. Treating this with chloroform and preparing it as a phage lysate usually works well.
  • P1 lysis is accelerated under reducing conditions (Ryland Young's Lab). Adding 1 mM DTT to the top agar allows P1 to develope better plaques. It follows that reducing agents may help the donor lysate develope and help the recipients infecected with infectious P1 to lyse before plating. If you're having trouble getting a high titer of donor phage, try β-mercaptoethanol at 1/1000 culture volume.


Who to ask about this protocol

Chris, Sean, Kathleen

Personal tools