Oct 17, 2024
Extraction of high molecular weight DNA from bacteriophage
This protocol is a draft, published without a DOI.
- Samuel Montgomery1,2,3,
- Mitchell Hedges1
- 1Telethon Kids Institute;
- 2University of Western Australia;
- 3Curtin University
Protocol Citation: Samuel Montgomery, Mitchell Hedges 2024. Extraction of high molecular weight DNA from bacteriophage. Protocol exchange https://protocols.io/view/extraction-of-high-molecular-weight-dna-from-bacte-dh7w39pe
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: August 01, 2024
Last Modified: October 17, 2024
Protocol Integer ID: 104406
Keywords: phage, bacteriophage, DNA extraction, DNA isolation, precipitation, zinc chloride, high molecular weight, HMW DNA, phage concentration
Funders Acknowledgement:
Medical Research Future Fund
Grant ID: 2023559
Disclaimer
The digestion step (where exogenous bacterial nucleic acids are degraded) may require optimisation i.e. longer incubation times or additional volume of enzyme. Sufficient removal of bacterial nucleic acids can be dependent on the growth media used for bacterial propagation, the growth characteristics of the host, the composition of the phage collection buffer and ultimately the yield of bacterial nucleic acid liberated during phage propagation.
Abstract
Extraction of High Molecular Weight DNA from dsDNA bacteriophages can be achieved using an isopropanol precipitation method which can be used for long read whole genome sequencing, such as Oxford Nanopore Technology.
This is a forked protocol from https://dx.doi.org/10.17504/protocols.io.3byl4qwqovo5/v1, which instead uses zinc chloride for bacteriophage virion precipitation to concentrate the sample.
Guidelines
This protocol can be used to extract DNA from bacteriophage within bacterial lysates.
It is ideal to extract phages which have a titre of 1 x 1010 to 1 x 1011 PFU/mL, however lower titres can be extracted successfully.
Materials
The following materials were utilised for this protocol:
- Puregene tissue kit (Qiagen, #158023) containing cell lysis solution, protein precipitation buffer, proteinase K, RNase A (4 mg/mL), and DNA hydration buffer
- Ethanol, molecular grade (Sigma, #E7023)
- UltraPure molecular grade water (ThermoFisher, #10977015)
- DNAse I (NEB, #M0303S)
- DNAse I 10x reaction buffer (NEB, #B0303SVIAL)
- Isopropanol, molecular grade (Sigma, #I9516)
- Zinc chloride (ZnCl2) powder (Merck, Cat#: Z0152-50G)
- Hydrochloric acid 32% AR Grade (Chem Supply, Cat# HA020-500M)
- DNA LoBind 2.0mL tubes (Thermo Fisher, #30108.078)
Safety warnings
While this protocol does not utilise common hazardous chemicals used for DNA extraction (phenol/chloroform), care should be taken to follow the recommendations in the MSDS provided which each reagent, and ensure proper storage for the dangerous goods utilised in this protocol (flammable liquids etc)
Before start
This protocol extracts DNA from a 700μL aliquot of bacteriophage lysate, however the starting lysate volume can be scaled up. If scaling up, adjust the reagent volumes in all steps up until the zinc chloride pelleting step, ensuring consistent ratios throughout the protocol to this point. Once the zinc chloride pellet has had the supernatant removed, the protocol volumes resume at the described amount.
Preparation of reagents
Preparation of reagents
17m
17m
Ethanol (70% v/v)
Create a 70 % (v/v) solution of molecular grade ethanol using UltraPure water (approx. 400 µL per sample)
2m
Digestion Master Mix
- Calculate the required volume of Digestion Mix for the number of samples you wish to process
- Each 700 µL sample of bacteriophage lysate requires 78 µL of Digestion Mix
- Create this by adding 1 µL of DNAse I and 1 µL of RNAse A to 76 µL of 10X DNAse I reaction buffer and place on ice
3m
Zinc chloride solution (2 Molarity (M) )
- Weigh 0.68 g of ZnCl2 and add to 2.5 mL of molecular grade water
- Carefully add 2.5 µL of 32 % (v/v) hydrochloric acid to the zinc solution and gently swirl to allow complete dissolution. Note: this step generates heat
- Measure a small aliquot of the solution on a pH meter, a 2 Molarity (M) solution of zinc chloride should have a pH 4.5 to 7 to ensure Zn+2 ions are available in solution
10m
EDTA Solution (0.1 Molarity (M) )
Add 200 µL of 0.5 Molarity (M) molecular grade EDTA to 800 µL of UltraPure water
2m
DNA extraction
DNA extraction
2h 8m
2h 8m
Add 78 µL of Digestion Master Mix to 700 µL of syringe filtered (0.22 µm ) bacteriophage lysate in a 1.5mL LoBind microtube and mix via gentle pipetting
1m
Incubate for 00:30:00 at 37 °C using a Thermomixer without shaking.
Note: longer incubation times may be necessary to achieve complete degradation of bacterial DNA and RNA. It is advisable to quantify the amount of bacterial DNA and RNA pre- and post-digestion to ensure bacterial nucleic acids do not contamination phage DNA.
30m
Add 2.5 % (v/v) of 2 Molarity (M) zinc chloride solution (freshly made) to the bacteriophage lysate for a final concentration of 50 millimolar (mM) and mix via gentle pipetting
1m
Incubate the sample for 00:05:00 at 37 °C
5m
Centrifuge for 00:10:00 at 8000 rcf, Room temperature and remove supernatant by aspiration
10m
Add 50 µL of 0.1 Molarity (M) EDTA solution to the phage pellet, pipetting and stirring gently with the pipette tip to break up the pellet, noting it is not necessary to resuspend the pellet during this step
1m
Add 1.5 µL of Proteinase K (20 mg/mL ) and 250 µL of Cell Lysis Solution to the phage pellet and mix by gently pipetting up and down 25 times
1m
Incubate for 01:00:00 at 56 °C in a thermomixer at 500 rpm and then pre-cool a centrifuge to 4 °C
1h
Place sample on ice for 00:01:00
1m
Add 100 µL of protein precipitation solution and mix thoroughly by inverting the tube 50 times
2m
Incubate samples on ice for 00:05:00
5m
Centrifuge tube at 16000 rcf, 4°C, 00:10:00
10m
The precipitated proteins should form a tight pellet. If the protein pellet is not tight, or there is still some in solution, repeat the incubation and centrifugation
Add supernatant to a new 1.5mL LoBind tube, careful not to disturb the protein pellet
1m
DNA precipitation
DNA precipitation
1h 46m
1h 46m
Add 300 µL of isopropanol to a new 1.5mL LoBind tube and carefully add the supernatant from the previous step by pipetting from the air-liquid interface the sample, mix by inverting the tube 50 times
3m
Incubate at Room temperature for 00:05:00
5m
Centrifuge at 16000 rcf, Room temperature, 00:05:00 placing the hinge of the tube at the top of the centrifuge to assist in identifying the location of the DNA pellet once centrifuged
5m
Discard the supernatant by slowly drawing with a pipette at the air-liquid interface to avoid disturbing the pellet, which may be difficult to see
Note
Pellets when precipitated with isopropanol will be near invisible, and easily detached from the tube, so care must be taken not to discard the DNA pellet.
1m
Add 300 µL of 70 % (v/v) ethanol to the sample and invert 10 times to wash the pellet
2m
Centrifuge at 16000 x g for 00:01:00
1m
Discard the supernatant, and allow the pellet to air dry for 00:05:00
5m
Add 30 µL of DNA hydration solution or nuclease free water to the pellet
1m
Incubate at 65 °C for 01:00:00 to dissolve the DNA, or incubate in the fridge at 4 °C Overnight
1h
After this step, DNA is ready to be quantified and used downstream. DNA resuspended in water should be stored at -20 °C , while DNA resuspended in DNA hydration solution can be stored for up to one month at 4 °C
Protocol references
Saladié M, Caparrós-Martín JA, Agudelo-Romero P, Wark PAB, Stick SM, O'Gara F. Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid. Front Microbiol. 2020 Oct 6;11:572504. doi: 10.3389/fmicb.2020.572504. PMID: 33123104