Viral purification from bacterial culture

Page last modified on: 2025-November-27

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Metadata

Title	Category	Adapated and used by	last accessed	Link to protocol	Primary origin for protocol
Viral purification from bacterial culture	Sample Preparation, Virus	EMBL, Sarah Schulz	2021-11-09	protocols.io	(Bachrach U., 1971)

Protocol

1.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

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

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

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

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

6.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

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

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

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

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

11.Store at 4°C

Source

In-house protocol provided by Sarah Schulz.

References