I am trying to infect E. coli with M13 phage (both purchased from NE Biolabs). I have been following the protocol but still cannot get plaques. I have listed the steps I have been following. I would appreciate any feedback. I am an undergrad, who took over a grad students project, and I have very little experience with microbiology. E. coli is kept at -80C and M13 at -20C.
1. Use frozen E. coli (Biolabs) to plate for single colonies.
2. Add single colony to LB broth, grow to approx OD600=0.4.
3. Dilute M13 (Biolabs) phage of 1 x 10^13 pfu/ml. (I have tried both PBS and LB broth, which Biolabs suggested)
Use microfuge tubes - Add 990 microliters of LB to 10 microliters of M13 to get 1 x 10^11 pfu
Then 100 microliters of that to 900 microliters of LB - 1 x 10^10......and cont until I get to 10^1.
4. LB Agar plates are warmed at 37C for an hour.
5. Top agar is prepared and kept between 45-50C.
6. Mix E. coli and M13 in microfuge tube (by flicking the tube). I have used various quanities, such as
1 ml E. coli : 50 microliters M13,
200 microliters E. Coli : 10 microliters M13 (Biolabs suggest in protocol)
Wait 5 min at RM temp.
7. Add E. coli/M13 mix to 3 ml of warm top agar. Vortex. Pour on LB plates. Allow to set. Invert and store at 37C overnight.
I have used to 10^1 through 10^7 m13 dilutions and so far my plates either look like an overgrown lawn of bacteria or dots of bacteria all over the plate. I have no clear areas that look like plaque. I would love to hear from anyone. Thank you