Mar 09, 2023
Virome DNA extraction with phenol-chloroform
- Adair Borges1
- 1Arcadia Science
- Arcadia Science
Protocol Citation: Adair Borges 2023. Virome DNA extraction with phenol-chloroform. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1oxdklr2/v1
Manuscript citation:
Borges A, Dutton R, McDaniel E, Reiter T, Weiss ECP. (2023). Paired long- and short-read metagenomics of cheese rind microbial communities at multiple time points. https://doi.org/10.57844/arcadia-0zvp-xz86
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: February 22, 2023
Last Modified: January 30, 2024
Protocol Integer ID: 77393
Keywords: phage, dna, extraction, phenol, chloroform, virus, virome, microbiome, microbial community, cheese, virome dna extraction with phenol, virome dna extraction, weight dna extraction from bacteriophage, weight dna extraction, virome, using phenol, chloroform this protocol detail, bacteriophage, extraction, phenol, single phage, dna, chloroform, microbial community
Abstract
This protocol details high-molecular-weight DNA extraction from bacteriophages using phenol-chloroform. This protocol can be performed on a virome harvested from a microbial community, or a single phage amplified in the lab.
Troubleshooting
Degradation of host nucleic acids
To degrade any non-encapsulated nucleic acids, treat the concentrated phage lysate with 10 µg/mL RNase A (NEB T3018) and 10 µg/mL DNase I (NEB M0303). Digest for 1 h at room temperature or 4 °C overnight. Store phage at 4 °C.
Phage DNA extraction (using phenol-chloroform)
Strip away phage capsids with 1 µl of Proteinase K (NEB P8107S) + 0.05% SDS. Incubate at 55 °C for 60 min on a Thermomixer at 250 rpm. If you only have a static heat block, vortex every 10 min.
In fume hood, add an equal volume of phenol – chloroform – isoamyl alcohol mixture, pH 8 (Sigma Aldrich 77618) to the digested phage. If the volume of phage is low, consider bringing up the volume to 500 µl with 1× TE buffer. Incubate at room temperature for 10 min, vortexing regularly to keep phenol mixed in.
Spin sample at maximum speed at 4 °C for 10 min.
In the fume hood, transfer the aqueous layer (top layer) to a new tube. Make sure to avoid the interface, and do not transfer any any debris or organics to the new tube. Add equal volume of chloroform to the sample and invert to mix.
Spin sample at maximum speed at 4 °C for 5 min.
Repeat steps 5 and 6, extracting the aqueous layer with more chloroform.
Transfer aqueous layer to new tube. You can perform all subsequent steps on the benchtop.
Isopropanol precipitation
Add a 10% volume of 3 M sodium acetate to the sample, and an equal volume of isopropanol. If you anticipate a low DNA yield, you can use glycogen (like GlycoBlue, ThermoFischer AM9515) as a coprecipitant at 50–150 µg/mL. Invert to mix, and move to 4 °C for 2 h (can be left overnight).
Spin sample at maximum speed at 4 °C for 30 minutes.
DNA will be a glassy smear or pellet on the side of the tube. Carefully pipette off and discard the liquid.
Wash pellet with 1 mL freshly made 70% ethanol (room temperature, not cold). DNA will start to turn white. Check to make sure the DNA isn’t stuck on the sides of the tube. If it is, wash it down with the 70% EtOH.
Spin sample at maximum speed at 4 °C for 5 min.
Carefully pipette off and discard the liquid, and then wash with 500 µl of 70% ethanol. Spin sample at maximum speed at 4 °C for 5 min.
Carefully pipette off and discard the liquid. Do short spins, and pipette with smaller and smaller pipette volumes until all the ethanol is removed. Let dry for 5 min or less.
Resuspend pellet in 30 µl of nuclease-free water. It can help to warm the sample to 50 °C. The volume of water can be adjusted if you anticipate a large DNA yield.
Use Nanodrop to assess contamination, and Qbit or Tapestation to quantify DNA yield.