Aug 08, 2025
Bacteriophage DNA extraction
- Madison Altieri1
- 1Bowling Green State University
Protocol Citation: Madison Altieri 2025. Bacteriophage DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodr99g4o/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: In development
We are still developing and optimizing this protocol
Created: August 08, 2025
Last Modified: August 08, 2025
Protocol Integer ID: 224264
Keywords: dna extraction for bacteriophage dna, bacteriophage dna extraction, genomic dna from other bacteriophage, dna extraction, bacteriophage dna, genomic dna, other bacteriophage, protocols step, phage, marijn ceelen at wageningen university
Abstract
This is a DNA extraction for bacteriophage DNA modified from Marijn Ceelen at Wageningen University for T7 E. coli phage. All protocols steps are the same, but detailed to how I have performed it to get genomic DNA from other bacteriophage. Thank you Marijn for supplying the original and saving my dissertation!
Materials
DNase I (Invitrogen REF 18068015)
EDTA (500mM/0.5M)
- 93g EDTA
- 500mL ddH2O
- Adjust pH to 8 with ~10g NaOH pellets
- Autoclave and store at room temperature
Proteinase K (Invitrogen REF 25530049)
SDS 10%
100mL ddH2O
10g SDS
Sodium Acetate (3M)
- dissolve 12.3g sodium acetate anhydrous in 40mL ddH2O
- adjust pH to 5 with glacial acetic acid
- bring to 50mL
Troubleshooting
Day 1
1d 1h 45m
Phage stock
Use 0.22µm filtered phage stock solution stored at 4˚C.
Transfer 1mL of phage stock (108+) into closed screw cap tube.
Degrade Bacterial DNA (optional RNA)
50 µL of DNase I 10X buffer
1µL DNase I (1U/µL)
Optional: 1µL 10mg/mL)
Flick tube
Incubate 01:30:00 at 37˚C water bath
1h 30m
Chelate DNase
Add 20µL of 0.5M EDTA pH 8.0
Flick tube
Incubate 00:15:00 t 75˚C water bath
15m
Break Capsid
Add 20µL of Proteinase K (20mg/mL)
Add 50µL 10% SDS
Flick tube
Incubate overnight (~24:00:00) - no shaking
1d
Day 2
2h 20m
Phenol:Chloroform:Isoamyl Alcohol
Add equal volume phenol:chloroform:isoamyl alcohol (25:24:1)
Invert tube to mix well - DO NOT VORTEX
Centrifuge at room temperature 00:10:00 at 10,000xg (9,500 RPM)
Carefully pipette off aqueous layer (top later) into a new tube
Make sure that you are using the appropriate plastics as chloroform melts some plastics. Eppendorf tubes are good to use.
10m
Chloroform:Isoamyl Alcohol
Add equal volume chloroform/isoamyl alcohol (24:1)
Invert tube to mix well - DO NOT VORTEX
Centrifuge at room temperature 00:10:00 at 10,000xg (9,500 RPM)
Carefully pipette off aqueous layer (top later) into a new tube
10m
Sodium Acetate
Add 1/10 volume of 3M Sodium Acetate
Flick tube
| Sample | Sodium acetate | |
| 1mL | 100µL |
100% Ethanol
Add 2.5x the volume of 100% Ethanol
Invert tube to mix well - DO NOT VORTEX
| Sample | 100% Ethanol | |
| 1mL | 2,750µL |
Incubate at -20˚C for 00:30:00 in -20˚C freezer
Centrifuge at room temperature 00:10:00 at 4,668xg (5,000 RPM)
Discard the supernatant: at this point, you should see a white(ish) pellet along the side and bottom of your tube. Do not disturb this pellet as you remove the supernatant. It is your DNA.
30m
70% Ethanol and Drying Pellet
Rinse pellet with 50-100µL of 70% ethanol -pipette mix to dissolve pellet - DO NOT VORTEX
Dry the pellet between 50-60˚C heat block (Optional: within a fume hood will go faster). Do not go above 70˚C as it reaches boiling point of ethanol (78˚C), and could damage the DNA.
1h 30m
Nanodrop
Make sure all ethanol is evaporated before adding MilliQ (MQ) water (Better option: Ultrapure DNase, RNase free water)
Add 32µL of MQ and pipette mix to dissolve pellet - DO NOT VORTEX