TITLE: Virome DNA extraction with phenol-chloroform
AUTHORS: Adair  Borges

[DESCRIPTION]
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.

[STEPS]
SECTION: Degradation of host nucleic acids
1. 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. 
SECTION: Phage DNA extraction (using phenol-chloroform)
2. 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.
SECTION: Phage DNA extraction (using phenol-chloroform)
3. 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.
SECTION: Phage DNA extraction (using phenol-chloroform)
4. Spin sample at maximum speed at 4 °C for 10 min. 
SECTION: Phage DNA extraction (using phenol-chloroform)
5. 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. 
SECTION: Phage DNA extraction (using phenol-chloroform)
6. Spin sample at maximum speed at 4 °C for 5 min. 
SECTION: Phage DNA extraction (using phenol-chloroform)
7. Repeat steps 5 and 6, extracting the aqueous layer with more chloroform. 
SECTION: Phage DNA extraction (using phenol-chloroform)
8. Transfer aqueous layer to new tube. You can perform all subsequent steps on the benchtop. 
SECTION: Isopropanol precipitation
9. 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). 
SECTION: Isopropanol precipitation
10. Spin sample at maximum speed at 4 °C for 30 minutes.
SECTION: Isopropanol precipitation
11. DNA will be a glassy smear or pellet on the side of the tube. Carefully pipette off and discard the liquid.
SECTION: Isopropanol precipitation
12. 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.
SECTION: Isopropanol precipitation
13. Spin sample at maximum speed at 4 °C for 5 min.
SECTION: Isopropanol precipitation
14. 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.
SECTION: Isopropanol precipitation
15. 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.
SECTION: Isopropanol precipitation
16. 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.
SECTION: Isopropanol precipitation
17. Use Nanodrop to assess contamination, and Qbit or Tapestation to quantify DNA yield.