Jun 17, 2026

HMW DNA extraction from Water Filters Using Magnetic Beads

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld6q19g5b/v1
  • 1Lawrence Berkeley National Laboratory;
  • 2Jorgmundir
Lauren Lui
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld6q19g5b/v1
Protocol Citation: Lauren Lui, Torben Nielsen 2026. HMW DNA extraction from Water Filters Using Magnetic Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6q19g5b/v1
Manuscript citation:
1. Lui, LM and Nielsen TN (2024). Decomposing a San Francisco estuary microbiome using long-read metagenomics reveals species- and strain-level dominance from picoeukaryotes to viruses. mSystems. 9 (9), e00242-24. https://doi.org/10.1128/msystems.00242-24

2. Lui, LM and Nielsen TN (2026) Contrasting population structures coexist in a strain-resolved estuarine microbiome
bioRxiv, https://doi.org/10.64898/2026.03.20.713316
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: October 06, 2025
Last Modified: June 17, 2026
Protocol  Integer ID: 229083
Keywords: DNA extraction, microbiome, bead-based, metagenomics, hmw dna extraction from water filter, hmw dna extraction, dna from biomass, dna shearing, read metagenomic, microbiome study, dna, water sample, biorxiv, using magnetic bead, purification, pes filter, carboxyl bead mixture, water filter, biomass, enzymatic lysi
Funders Acknowledgements:
United States Department of Energy
Grant ID: DE-AC02-05CH11231
Abstract
This protocol provides a methodology for extracting high-molecular weight (HMW) DNA from biomass from water samples collected on PES filters and is designed to support microbiome studies requiring strain-level resolution. Developed for applications such as long-read metagenomics, we describe gentle handling techniques to prevent DNA shearing. We outline a detailed procedure spanning sample storage, enzymatic lysis, and purification using a carboxyl bead mixture. This specific protocol was featured in research published in mSystems in August 2024 and a preprint posted on bioRxiv. Please cite our papers and this protocol if you use this method or felt inspired by it in your experimental methodology.
Materials
Sample
  • Biomass from water samples collected on PES filters. The optional part of this protocol only works with PES filters because PES dissolves in chloroform.

Materials
  • 5mL and 1.5mL low retention tubes
  • Vortemp or incubator
  • Hula mixer or other agitator for magnetic bead binding
  • Magnetic rack that can hold 5mL tubes, magnetic racks for 15mL tubes may work.
  • Optional: phase lock tubes for separating chloroform from lysis buffer

Reagents
  • Optional: Chloroform
  • 70-80% Ethanol (10-15mL per sample)
  • QIAGEN Proteinase K
  • QIAGEN RNase A
  • Lysozyme 100mg/mL

Lysis Buffer
  • 30mM Tris-HCL
  • 30mM EDTA
  • 800 mM Guanidine HCL
  • 0.5% Triton X-100
  • 0.5% Tween-20

Carboxyl Bead Mixture (See appendix for recipe)
  • 1 M Tris pH 8
  • dH2O
  • Sera-Mag SpeedBeads Carboxyl Magnetic Beads Hydrophilic Carboxylic acid content: 0.40 - 0.58 meq/g (VWR catalog #10204-670, Cytiva part #65152105050250)
  • 5M NaCl or NaCl in powder form
  • PEG-8000
  • 0.5M EDTA
  • Tween-20
Troubleshooting
Problem
I have large filters (e.g. 142mm). I can't fit these into 5mL tubes.
Solution
We have used sterile scissors to cut up the filters and put them into 50mL low retention tubes with 18mL of lysis buffer. Small filter pieces may end up in the final elution. Scale up the enzymes accordingly (Proteinase K 450 uL, RNase A 360 uL, Lysozyme 180 uL). The amount of bead mixture will be increased to 18mL. For the ethanol cleaning step, you can choose to split the sample into 3mL portions and use 5mL tubes, or split the sample in half and clean up in 15mL tubes. Yield and purity are not affected.
Safety warnings
If you choose to use chloroform, read about safe handling of this chemical. Only use in the fume hood with appropriate PPE.
Before start
Tips on Handling and Storage of HMW DNA
  • Note that there are also tips on how to handle high-molecular weight (HMW) DNA in the back of the LISA Workshop handbook that we ran in December 2023.
  • If you haven’t handled HMW DNA before, try extracting DNA from a bacterial isolate first and try running it out on a gel to check for DNA degradation. Note that if you overload the gel, HMW DNA will tend to become streaky and stick in the wells of the gel. If the DNA is degraded, then you may be handling the DNA too roughly or there is something in the sample that is degrading the DNA
  • Extracting HMW DNA from environmental samples tends to be different because the cells may not have been actively growing and much of environmental DNA tends to be short fragments. The DNA is generally more fragmented and damaged.
  • Handling HMW DNA is different from how we are usually trained to handle DNA and requires a conscientious approach.
  • Pipetting up and down vigorously and vortexing for long periods of time (>10 s) will definitely shear the DNA. Pipetting slowly or with wide bore tips is recommended. Despite this, don't be too paranoid about handling the DNA unless you're trying to get ultra-long reads.
  • Using lo bind tips can help with more accurate pipetting of HMW DNA as it can be more viscous.
  • If diluting HMW DNA, wait at least 10-20 minutes before measuring the concentration as it takes time for it to resuspend. If you measure the concentration before the DNA fully resuspends, the measurement will be lower than actual.
  • We recommend reading the NEB Monarch HMW DNA Extraction Kit for Tissue Manual (NEB #T3060S/L) for tips on handling HMW DNA, especially for resuspension and storage tips. HMW DNA can form tangles and become difficult to fully resuspend.
  • Store in 4C for use within a few days to 2 weeks if high quality (260/230 >1.8, 260/280 > 1.8). If quality is low, freeze the sample to prevent degradation. Although freeze-thaws can fragment the DNA, impurities will also fragment the DNA if not frozen. Long term storage at -20C.
  • Storage in Tris-EDTA is recommended to reduce exposure to DNases.
Prep filters
After collecting biomass on the filter, place filters into lention tubes with biomass facing inward, away from the wall of the tube. This may require twisting the filter into a roll. Usually we use 47mm PES PES filters and put them into 5mL low retention tubes.

If filters are being stored for later, freeze at -20 C or -80 C. Avoid freeze-thaw cycles as this will damage the DNA.
Lysis
1h
Add 3 mL of lysis buffer to the filter.

Note
If filters were stored frozen, it is important to put lysis buffer on them as soon as possible after taking them out of the freezer.

Add 30 µL of lysozyme 100 mg/mL . Pulse vortex or invert to mix. Incubate 37 °C for 00:30:00 , preferably with shaking (e.g., Vortemp at 1000 RPM).

30m
Add 75 µL QIAGEN Proteinase K. Pulse vortex or invert to mix. Incubate 50 °C for at least 00:30:00 , preferably with shaking (e.g., Vortemp at 1000 RPM).
30m
Add 60 µL QIAGEN RNase A, and pulse vortex or invert to mix.
Incubate Overnight at 40 °C , preferably with shaking (e.g. Vortemp at 1000 RPM). Wrapping the top of the tube with parafilm is recommended to prevent spillage.


Note
We have added all enzymes at the same time before and incubated at 40C overnight with similar results. The longer lysis time is critical.


Transfer lysis buffer to new 15 mL or 5 mL low retention tube.
Pour the majority of buffer into a new low retention tube. Spin down briefly to concentrate the remaining liquid. For the remaining liquid use a pipet. Use wide bore tips or pipet slowly to preserve HMW DNA. If the DNA is in a ball, vortexing for 5 seconds is okay to break up the tangle and get it resuspended. Avoid over-vortexing as this will shear the DNA.
(Optional) Get DNA off the filter.
Add 1-2 mL of lysis buffer to the filter.
Add 3-4 mL chloroform to the filter.
The filter should dissolve. If it forms a little ball, then you need to add more chloroform.
Use a phase lock tube (optional) and centrifuge to obtain the supernatant and add to rest of the DNA.
Cleanup and concentration
10m
(Optional) Use a Qubit measurement on the lysis to estimate the amount of DNA. This will help you determine how much volume to elute in later. However, DNA loss during the cleanup process will vary and depend on the sample.
Add bead mixture (see Materials for recipe) to saved lysis at a 1 :1 ratio.

Mix gently by inversion for 5-15 minutes. We use a hula mixer. DNA will bind to beads.

Note
At this ratio of beads to lysis, small DNA fragments will be depleted (< 2000bp), so do not expect to get 100% recovery. Removal of these small fragments is helpful for increasing the yield from a Nanopore flow cell.


Place on magnetic rack to wash sample.
After the beads bind to the magnet, remove the supernatant.
Wash 3x with 70-80% ethanol. Do not spray beads directly with ethanol. This will knock the DNA off the beads. Make sure that the beads get covered with ethanol. This is usually at least 4-5mL of 70-80% ethanol. The more ethanol used, the cleaner the DNA will be. After adding the ethanol, close the tube and invert 1-2 times to wash the cap.
(Optional) After the final wash, spin the tube gently in a microfuge and then place back on the magnetic rack to pool remaining ethanol. Pipet off remaining ethanol.
Let the beads dry for 30 s to 1 minute to allow ethanol to evaporate, but do not allow the beads to become matte and crack. This will damage the DNA. Downstream nanopore applications are okay with some ethanol, so it is better to move to the resuspension step with some ethanol instead of dry beads.
Add 100uL TE or in volume based on initial DNA measurement and desired concentration. Resuspend beads and incubate at Room temperature or 37 °C for 00:10:00 . Incubation at 37C may help recover longer fragments.

10m
Pellet beads on magnetic rack.
Remove supernatant and put into a 1.5mL low retention tube.
Store the DNA at 4 °C for temporary storage or-20 °C for long term storage.

QC
Measure concentration of DNA on a Qubit. Wait at least 10-20 minutes until after resuspending the DNA before taking the measurement to improve accuracy.
You may wait to do the following QC steps if you choose to do size selection.
Look at quality on a nanodrop. Ideally we want to see 260/230 and 260/280 values >2.0, but >1.8 is ok. Lower values will reduce yield in Nanopore and Pacbio sequencing, or indicate contaminants that can damage the DNA over time.
Optionally, run the DNA out on a 0.5% gel, Tapestation, or Femtopulse to look at the fragment distribution. For directions on how to run the gel, look at the lab notebook of our LISA workshop: https://sites.google.com/lbl.gov/lisaworkshop/workshop-materials?authuser=0
Appendix: Carboxyl Bead Mixture
In a 50 mL conical using sterile stock solutions, prepare TE.
  • 500 uL 1 M Tris pH 8
  • 100 uL 0.5 M EDTA
  • fill conical to 50 mL mark with dH2O
Wash Sera-mag SpeedBeads
Resuspend Sera-mag SpeedBeads by vortexing and transfer 1 mL to a 1.5 mL microtube.
Place SpeedBeads on magnet stand until beads are drawn to magnet.
Remove supernatant with P200 or P1000 pipettor.
(Wash 1) Add 1 mL TE to beads, remove from magnet, mix, return to magnet.
Remove supernatant with P200 or P1000 pipettor.
(Wash 2) Add 1 mL TE to beads, remove from magnet, mix, return to magnet.
Remove supernatant with P200 or P1000 pipettor.
Add 1 mL TE to beads and remove from magnet. Fully resuspend and set microtube in rack (i.e. not on magnet stand).
Add 9 g PEG-8000 to a new 50 mL, sterile conical.

Add 10 mL 5 M NaCL (or 2.92 g) to conical.

Add 500 µL 1 M Tris-HCL to conical.

Add100 µL 0.5 M EDTA to conical.

Fill conical to ~ 49 mL using sterile dH2O. You can do this by eye, just go slowly.
Mix conical for about 3-5 minutes until PEG goes into solution (solution, upon sitting, should be clear).
Add 27.5 µL Tween-20 to conical and mix gently.

Resuspend 1 mL SpeedBead + TE solution by vortexing briefly and transfer to 50 mL conical.
Fill conical to 50 mL mark with dH20 (if not already there) and gently mix 50 mL conical until beads are resuspended.
(Optional) Test against AMPure XP using aliquots of ladder
See example and explanation here: https://www.keatslab.org/blog/pcrpurificationampureandsimple
Wrap in tinfoil (or place in dark container) and store at 4°C. Beads are paramagnetic and will lose their magnetism over time, similar to Ampure beads.
Protocol references
References 1 and 2 are where this protocol has been used. The rest of the references are what we drew from to develop this protocol. We were unable to find a DOI for reference 4, but it is based on reference 5.

1. Lui, LM and Nielsen TN (2024). Decomposing a San Francisco estuary microbiome using long-read metagenomics reveals species- and strain-level dominance from picoeukaryotes to viruses. mSystems. 9 (9), e00242-24. https://doi.org/10.1128/msystems.00242-24

2. Lui, LM and Nielsen TN (2026) Contrasting population structures coexist in a strain-resolved estuarine microbiome
bioRxiv, https://doi.org/10.64898/2026.03.20.713316

3. Carvalho, R., Kellogg, C., & Lemay, M. Serapure beads preparation and testing (Version 1). protocols.io. https://dx.doi.org/10.17504/protocols.io.3byl49952go5/v1

4. Faircloth, B., & Glenn, T. (2011, November 19). Serapure v2.2. Department of Ecology and Evolutionary Biology, UCLA. https://ethanomics.wordpress.com/wp-content/uploads/2012/08/serapure_v2-2.pdf

5. Rohland, N., & Reich, D. (2012). Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Research, 22(5), 939–946. https://doi.org/10.1101/gr.128124.111

6. Oberacker, P., Stepper, P., Bond, D. M., Höhn, S., Focken, J., Meyer, V., Schelle, L., Sugrue, V. J., Jeunen, G.-J., Moser, T., Hore, S. R., von Meyenn, F., Hipp, K., Hore, T. A., & Jurkowski, T. P. (2019). Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLOS Biology, 17(1), e3000107. https://doi.org/10.1371/journal.pbio.3000107

7. rute.carvalho Carvalho, Colleen Kellogg, Matt Lemay 2024. Serapure Beads Preparation and Testing. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49952go5/v1