Day 2
1) The next day, transfer 50µl of the overnight into 2 ml of LB amp. Shake at 37°C for
about 3 hours, until OD600 is about 0.7.
2) Add 25µl of helper phage (109 phage for an moi of about 10). Shake at 37°C for 1
hour. (The helper phage we use is VCS-M13, available from Stratagene.)
3) Dilute 400µl into 10 ml of LB amp kan, and shake at 37°C overnight. (Phage is
kanR)
Day 3
1) Pellet cells in eppendorfs (1.5ml per tube)/10 min.
2) Carefully remove only 1 ml of the supernatant to a new set of tubes.
3) Add 400µl 20% PEG 8000, 2.5M NaCl. Vortex and leave at RT for 15 minutes.
4) Pellet phage/10 minutes in microfuge. A loose pellet should be visible.
5) Resuspend each pellet in 100µl TE, and combine into two tubes. Phenol/chloroform
extract and ethanol precipitate ss DNA.
6) Resuspend each pellet in 100µl TE and run 3µl on a gel.