In order for M13 phage to be efficiently amplified, it is critical that cultures be well aerated, and that cultures be infected early in their growth phase. We recommend amplification in 20 mL cultures in 250 mL Erlenmeyer flasks, in a shaker set to 250 rpm. Amplification in smaller vessels, such as 50 mL conical tubes, will result in much lower yields of amplified phage. M13 phage should either be added to an early-log culture, A600 < 0.01, or to a 1:100 dilution of an overnight culture. Yield of amplified phage is maximal after 4.5-5 hours at 37°C; longer incubation may result in deletions and is not recommended. If carrying out nonspecific elution with pH 2.2 glycine buffer, the eluted phage must be neutralized as described in the Manual prior to amplification.