Phage DNA Extraction Protocol

Version not yet assigned, updated 2026-09-30. https://dustinedwards.info/research/protocols/phage-dna-extraction

Updated: 2026-09-30
First used: fall 2018

Reagents

Reagent	Stock	Final	Amount	For 5 tubes	Notes
high-titer lysate			5 ml		Filter-sterilized and titered first. How to make one is on the phage isolation protocol page.
nuclease mix (DNase I plus RNase A)			20 µl per 5 ml		20 µl per 5 ml of lysate. The lab's protocol names it only as DNase I plus RNase A. The Phage Discovery Guide gives a recipe in its reagent recipes.
ZnCl2	2 M	40 mM	20 µl per tube	100 µl	2 M ZnCl2, 20 µl per 1 ml of lysate. That is 40 mM final, the concentration Santos (1991) gives.
TES buffer			500 µl per tube	2,500 µl	0.1 M Tris-HCl, pH 8; 0.1 M EDTA; 0.5% SDS (the lab's protocol as copied into its notebooks from 2018 on). Recipe below.
proteinase K	10 mg/ml or 20 mg/ml	50 to 100 µg/ml	1.25 to 5 µl per tube	6.25 to 25 µl	50 to 100 µg/ml final. The lab has used two stocks. Its protocol, as copied into notebooks in 2018, 2019 and 2021, lists 10 mg/ml. The Finny genome announcement (2019) reports 20 mg/ml, which is also the stock the Phage Discovery Guide lists, as a 2023 notebook copied it. Check the label on your tube, and use the volume for that stock in step 6.
potassium acetate (pH 5.2)	3 M		60 µl per tube	300 µl	3 M, pH 5.2.
isopropanol	100% or 80%		500 µl per tube	2,500 µl	500 µl per tube, at room temperature. The lab has used both 100% and 80% isopropanol.
ethanol	70%		250 µl per wash		70%, at room temperature, 250 µl per wash, two washes.
nuclease-free water			50 µl		Not Tris or TE: the Phage Discovery Guide specifies nuclease-free water for DNA sent for sequencing.
sodium acetate (Rescue)	3 M		0.1 volume
sodium chloride (Rescue)		0.2 M			In place of sodium acetate if SDS may remain.
ice-cold ethanol (Rescue)	100%		2.5 to 3 volumes
ice-cold 75% ethanol (Rescue)	75%		0.5 ml
glycogen carrier (optional) (Rescue)	20 mg/ml		1 µl

TES buffer

Tris-HCl (pH 8), 0.1 M final
EDTA, 0.1 M final
SDS, 0.5% final

Equipment

microcentrifuge tubes; microcentrifuge; heat block; NanoDrop; Qubit 3.0

Part A: collect the phage and open the capsids

Add 20 µl of nuclease mix to 5 ml of high-titer lysate. Incubate at 37 °C for 10 minutes.
Split the lysate into 5 microcentrifuge tubes of 1 ml each.
Add 20 µl of 2 M ZnCl2 to each tube. Incubate at 37 °C for 5 minutes.
Spin at 10,000 rpm for 1 minute. Remove the supernatant quickly and keep the pellet.
Resuspend each pellet in 500 µl of TES. Incubate at 60 °C for 15 minutes.
Troubleshooting: The zinc chloride pellet does not give DNA.
Add proteinase K to 50 to 100 µg/ml. Check the tube's label for the stock: 2.5 to 5 µl per tube of a 10 mg/ml stock, or 1.25 to 2.5 µl of a 20 mg/ml stock. Incubate at 55 to 60 °C for 30 to 60 minutes.
Pause point.
In this lab, the prep has been paused overnight at 4 °C after this step.

Part B: remove the protein

Add 60 µl of 3 M potassium acetate (pH 5.2). Mix hard, until the precipitate is fluffy and very white, then put the tube on ice for 15 minutes.
Critical step.
Mix until the precipitate is fluffy and very white before the ice. A mix that set solid gave a run of salty preps.
Troubleshooting: The mix sets solid on adding potassium acetate, and the preps come out salty.
Spin at 12,000 rpm for 1 minute at 4 °C. Move the supernatant to a fresh tube; the pellet is protein. Spin the supernatant again for 3 minutes and move it again, so no precipitate is carried into the isopropanol.
Troubleshooting: Salt in the final DNA: a large A230 peak and a low A260/230.

Part C: precipitate and wash the DNA

Add 500 µl of room-temperature isopropanol to the supernatant and mix. Leave it at room temperature for about 10 to 30 minutes. Do not put it on ice or leave it overnight.
Critical step.
Keep the isopropanol at room temperature. Salt also precipitates in isopropanol, and salt carried into the DNA was this method's most common failure.
Spin at top speed for 15 to 30 minutes. Discard the supernatant and keep the pellet.
Wash the pellet with 250 µl of room-temperature 70% ethanol and spin at top speed in a microcentrifuge for 5 to 15 minutes. Discard the ethanol.
Spin: 10,000 to 15,000 x g
Troubleshooting: Very low yield and A260/230, with pellets too salty to dissolve.
Wash a second time the same way.

Part D: dry, dissolve and measure

After the last wash, pulse-spin and take off the last drops with a pipette. Air-dry for about 5 to 20 minutes, until the pellet turns clear. Do not dry for hours: overdried DNA redissolves poorly (QIAGEN).
Dissolve the pellet in nuclease-free water, as the Phage Discovery Guide specifies for DNA sent for sequencing. Dissolve the first pellet in 50 µl, then carry that same 50 µl to the second pellet, and so on through all 5, so the DNA from the five tubes ends up in one tube. Do not substitute Tris or TE.
Troubleshooting: A white, viscous "pellet" that soaks up the 50 µl of water. One read 100 ng/µl on the NanoDrop and failed PCR prep.
Measure the DNA. See checking DNA quantity and quality.

Rescuing salty or dilute DNA

Measure the DNA volume with a pipette: dial it down until the air gap disappears. Base the volumes on this volume, not on the ng/µl reading.
Add salt: 0.1 volume of 3 M sodium acetate (0.3 M final). If SDS may still be in the sample, use sodium chloride at 0.2 M final instead. Then add 2.5 to 3 volumes of ice-cold 100% ethanol. For 44 µl of DNA, that is 4.4 µl of sodium acetate and 110 to 132 µl of ethanol. For a very small amount of DNA, 1 µl of 20 mg/ml glycogen can be added as a carrier.
Hold at -80 °C for 1 hour to overnight, or at -20 °C overnight. One hour at about -16 °C did not work.
Spin at full speed at 4 °C for 30 minutes.
Wash twice with 0.5 ml of ice-cold 75% ethanol, with 10 minute spins at 4 °C.
Air-dry until the pellet turns clear, as in step 13.
Dissolve the DNA in 50 µl of nuclease-free water, as in step 14.

Troubleshooting

Step	Problem	Possible reason	Solution
7	The mix sets solid on adding potassium acetate, and the preps come out salty.	The acetate precipitate was not mixed to a fluffy consistency before the ice.	Shake it until the precipitate is gel-like and very white, then 15 minutes on ice. In one lab this fixed a run of salty preps (2,467.7 ng/µl, A260/280 2.06, A260/230 2.24).
8	Salt in the final DNA: a large A230 peak and a low A260/230.	Potassium acetate precipitate carried into the isopropanol.	Spin the potassium acetate step twice, as in step 8, so no precipitate is carried into the isopropanol.
9 to 12	Very low yield and A260/230, with pellets too salty to dissolve.	No ethanol wash. One group failed five times running without it (5.7 to 21.5 ng/µl, A260/230 0.03 to 0.07).	Room-temperature isopropanol and two room-temperature 70% ethanol washes, as in steps 9 to 12.
5	The zinc chloride pellet does not give DNA.	It was resuspended in phage buffer instead of TES.	Use TES, not phage buffer. The notebook marks phage buffer as never to be done.
13 to 14	A white, viscous "pellet" that soaks up the 50 µl of water. One read 100 ng/µl on the NanoDrop and failed PCR prep.	It is salt, not DNA.	Clean it up with the rescue, or seed fresh webbed plates from the ~100 µl of lysate kept in reserve and make another prep.