Viral purification from bacterial culture

Centrifuge 40 ml of bacterial culture infected with phage at 6000 g for 30 min, remove the supernatant and filter it through a 0.45 μm syringe filter

Centrifuge the filtrate at 35 000 g for 4 h, remove the supernatant and re-suspend the pellet in 600 μl SM buffer

Add 2 μl of DNAse I and 20 μl of 10x DNAse buffer and incubate at 37°C for 1.5 h

Incubate sample at 65°C for 30 min to inactivate DNAse I

Add 10 μl of 20 % SDS and 40 μl of Proteinase K (20 mg/ ml) and incubate at 37°C for 1 h

After the incubation, mix the sample with an equal amount of phenol:chloroform:isoamyl pH 8.0 (25:24:1) alcohol in a phase lock gel light tubes and centrifuge at 12 000 g for 5 min

Add 600 μl more of the phenol:chloroform:isoamyl alcohol to the tube and centrifuge at 12 000 g for 5 min

Transfer the aqueous phase to a new tube and add 1200 μl cold 100% ethanol

Incubate sample overnight at -80°C, and then centrifuge at 16 000 g for at 4°C for 1 h

Remove the supernatant and re-suspend the pellet in 100 μl TE buffer

Store at 4°C