Viral purification from bacterial culture
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Protocol
- 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
Source
In-house protocol provided by Sarah Schulz
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Citation
National Research Data Infrastructure for Microbiota Research (NFDI4Microbiota). (2024, September 30). Viral purification from bacterial culture. NFDI4Microbiota Knowledge Base.