Protocol Citation: sarah.schulz 2023. Viral purification from bacterial culture. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjq7wlmk/v1
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: March 06, 2023
Last Modified: March 21, 2023
Protocol Integer ID: 78214
Keywords: phage purification, phage
Abstract
Protocol for the purification of viral particles from bacterial liquid culture
Materials
50 ml Falcon tube, 0.45 μm syringe filter & syringe, SM buffer, DNAse I, 10x DNAse buffer , heatblock, 20 % SDS , Proteinase K, phenol:chloroform:isoamyl , phase lock gel light tubes , 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