My lab has been having issues re-amplifying a M13 phage library, which was donated to us from another lab. It yields binders that we have since validated, so there's no issue with the library itself but re-amplification yields are always low. We expect 10*13 but are seeing 10*10. We've gone through the normal troubleshooting steps (see below) with no luck.
There is a significant level of helper M13K07 phage contamination in our library phage stock (~1:100 CFU). I know that some cross contamination is almost inevitable given how many of us in the lab are using phage at once, but I'm wondering if this helper contamination is high enough that it's affecting our amplifications. High helper MOI can impair phage production due to loss of F pili (DOI: 10.1016/0042-6822(73)90131-1) so if we have preexisting helper in our cultures then when we add helper at a MOI of 10:1 we could actually be at a much higher MOI and could be losing pili.
Thoughts? Do you know what level of helper contamination in a library is tolerated by the F pili?
Basic amplification protocol:
- grow omnimax in 2YT to OD (check for contamination)
- infect with M13 library 30min shaking 37oC
- infect with M13K07 helper at MOI 10:1 for 1hr shaking 37oC
- scale up to final growth volume and grow overnight shaking 37oC
- spin down cells and
a. if testing amplification success, titre phage from supernatant
b. if preparing phage for use, double precipitate with PEG 8000 NaCl 20 min on ice, then titre
Unsuccessful troubleshooting:
- vary OD600 at time of infection (0.4-0.9)
- vary incubation rpm (150-220)
- vary growth volume (0.5L - 1L in a 2.8L baffled flask)
- remade all solutions from scratch
- fresh cells (omnimax)
if I am reading your question correctly, there is helper phage contamination at a level of 1:100 (ie 1%). When you are amplifying you are adding helper at MOI of 10. So unless I am misreading, I can't imagine there is enough helper in the stock that would be more than the MOI of 10 you are adding.
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