Dec 09, 2021

Public workspaceVLP Extraction from Fecal Samples V.2

DOI
dx.doi.org/10.17504/protocols.io.b2qaqdse
  • 1Copenhagen University
  • FOOD Micro UCPH
Frej Larsen
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DOI: dx.doi.org/10.17504/protocols.io.b2qaqdse
Protocol Citation: Frej Larsen 2021. VLP Extraction from Fecal Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.b2qaqdseVersion created by Frej Larsen
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: December 08, 2021
Last Modified: December 09, 2021
Protocol Integer ID: 55778
Abstract
This protocol is for isolation of bacteriophages from fecal matter. It is based on the protocol used for the COPSAC10 cohort and was originally described by Ling Deng in doi:10.3390/v11070667. Extraction is performed using centrifugation and ultrafiltration to isolate viral particles and get rid of contaminants.
Guidelines
This protocol is designed for extraction of bacteriophages from fecal samples. The sample should be 50-500mg of fecal material diluted in buffer (2xSM buffer for instance). Sample sizes outside these limits have not been tested and correct sequencing results can not be guaranteed.
Materials
Equipment:
  • Pipettors
  • Shaking table
  • Refrigerated centrifuge
  • Ice box

Consumables:
  • Crushed ice
  • 15 ml centrifuge tubes
  • 10 ml pipettes
  • 0.45 um PES filters
  • SM buffer (200 mM NaCl, 10 mM MgSO4, 50 mM Tris-HCl, pH 7.5)
Before start
Fill bucket with crushed ice

Turn on centrifuge and start the fast temp program to reach 4◦ C

Cool SM buffer in ice bucket
Thaw frozen sample in ice bucket
Transfer up to Amount500 mg of sample to centrifuge tube and add SM buffer to reach a final volume of Amount5 mL . Place centrifuge tube back in ice box

Homogenize samples by placing ice box on shaking board at Shaker300 rpm, 00:10:00

Centrifuge samples atCentrifigation5000 rcf, 4°C, 00:30:00

30m
Filter the supernatant through a 0.45 um PES filter into a new centrifuge tube
Transfer Amount2.5 mL of the sample to the outer chamber of the concentrator

Centrifuge sample in the concentrator at Centrifigation1500 rcf, 4°C, 00:20:00

20m
Pour off liquid from the inner chamber
Repeat the three previous steps until all liquid from the centrifuge tube has passed through the concentrator and the there is no liquid left in the inner chamber
Add 100 ul SM buffer to the inner chamber and centrifuge Centrifigation1500 rcf, 4°C, 00:02:00

2m
Leave in fridge to release VLPs from filter for at least 12 hours and at most for 1 week before proceeding with spotting or DNA/RNA extraction