Apr 11, 2025

Public workspaceViral purification from bacterial culture

 Forked from Viral purification from bacterial culture
This protocol is a draft, published without a DOI.
  • 1EMBL;
  • 2NFDI4Microbiota
  • NFDI4Microbiota
Maja Magel
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Protocol Citation: Sarah Schulz 2025. Viral purification from bacterial culture. protocols.io https://protocols.io/view/viral-purification-from-bacterial-culture-d7jh9kj6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2025
Last Modified: April 11, 2025
Protocol Integer ID: 126281
Keywords: phage purification, phage
Disclaimer
NFDI4Microbiota (https://nfdi4microbiota.de/) makes an effort to collect relevant and updated Standard Operating Procedures from active community members who frequently use or have recently used this protocol for their own experiments.
More information on microbiological FAIR issues can be found in our Knowledge Base (https://knowledgebase.nfdi4microbiota.de/Getting-Started/01-introduction.html).

Abstract
Protocol for the purification of viral particles from bacterial liquid culture.

Materials
Equipment
  • heatblock

Material
  • 50 ml Falcon tube
  • 0.45 μm syringe filter & syringe
  • phase lock gel light tubes

Reagents
  • SM buffer
  • DNAse I
  • 10x DNAse buffer
  • 20 % SDS
  • Proteinase K
  • phenol:chloroform:isoamyl
  • TE buffer


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
Protocol references
Source Protocol/ Primary Origin for Protocol:
Bachrach U, Friedmann A.1971.Practical Procedures for the Purification of Bacterial Viruses. Appl Microbiol22:.https://doi.org/10.1128/am.22.4.706-715.1971

Acknowledgements
Adapted and used by Sarah Schulz.
Last accessed: 2021-09-21