Jun 03, 2026

1. HMW Bacterial DNA Extraction Using NEB Monarch® HMW DNA Extraction Kit for Tissue

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5j8m6l1b/v1
  • 1CFSAN/FDA;
  • 2USDA;
  • 3US FDA
Jae Hee Jang
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5j8m6l1b/v1
External link: https://www.neb.com/protocols/2020/10/27/protocol-for-high-molecular-weight-dna-hmw-dna-extraction-from-bacteria
Protocol Citation: Jae Hee Jang, Kathryn Judy, Ellie Meeks, Maria Hoffmann 2026. 1. HMW Bacterial DNA Extraction Using NEB Monarch® HMW DNA Extraction Kit for Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5j8m6l1b/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: Working
We use this protocol and it's working
Created: April 24, 2023
Last Modified: June 03, 2026
Protocol  Integer ID: 80977
Keywords: DNA extraction, NEB, HMW, Bacteria, hmw bacterial dna extraction, monarch hmw dna extraction kit for tissue, monarch hmw dna extraction kit, hmw bacterial dna, extraction protocol for bacteria, extracted dna, purified hmw gdna, hmw dna, extraction protocol for tissue, purified dna, extracting high molecular weight, intact genomic dna from various tissue, longer dna into the mb range, protein removal step, optimized extraction protocol, intact genomic dna, modified extraction protocol, complete removal of rna, lysozyme for the efficient lysi, dna size, bacteria, dna onto the surface, longer dna, read genome assembly, gdna, bacterial cell wall, purity ratios for tissue, lysozyme
Abstract
The Monarch HMW DNA Extraction Kit for Tissue provides a rapid and reliable process for extracting high molecular weight (HMW), intact genomic DNA from various tissues and bacteria, as well as other sample types including yeast, insect, and amphibian. The optimized extraction protocol for tissue utilizes pestle homogenization and proteinase K digestion with agitation for sample lysis, followed by a protein removal step and precipitation of the extracted DNA onto the surface of large glass beads. A slightly modified extraction protocol for bacteria utilizes lysozyme for the efficient lysis of the bacterial cell wall prior to proteinase K digestion. DNA size ranges from 50 – ≥ 500 kb for the standard protocols and can be adjusted to produce longer DNA into the Mb range for soft organ tissues and bacteria. Purified DNA is recovered in high yield with excellent purity, including nearly complete removal of RNA. For tissue and bacteria, the processing time is ~90 minutes. Purity ratios for tissue and bacteria are typically 1.8-1.9 (A260/A280) and 2.1-2.5 (A260/A230). Purified HMW gDNA is suitable for a variety of downstream applications including long-read sequencing (Oxford Nanopore Technologies® and Pacific Biosciences®), optical mapping (Bionano Genomics®), and linked-read genome assembly (10X Genomics®).
Guidelines
  • Pay careful attention when pipetting and moving around samples to avoid chances of cross contamination.
  • HMW DNA extracted using this protocol will typically have a viscous, "stretchy" consistency; if the end product has the consistency of distilled water, then it is most likely that the lysis did not work.
Materials
Reagents
  • NEB Monarch® HMW DNA Extraction Kit for Tissue (NEB Cat# T3060L, 50 samples)
  • ≥95% Ethanol, Molecular Biology Grade (Fisher Scientific Cat# BP2818100 or equivalent)
  • 1x PBS (Thermo Fisher Scientific Cat# 10010023 or equivalent) Can be substituted with TE or Tris buffer.
  • Isopropanol, Molecular Biology Grade (Fisher Scientific Cat# 17140576 or equivalent)
  • for Gram-negative bacteria, Lysozyme (25 mg/mL) (MP Biomedicals Cat# 100831 or equivalent)
  • for Gram-positive bacteria, MetaPolyzyme Multilytic Enzyme Mix (Sigma Aldrich Cat# MAC4LDF)
  • Qubit 1x dsDNA Broad Range Kit (Thermo Fisher Scientific Cat# Q33266 or Q33265)

Supplies
  • Serological Pipet, 1 mL, Individually Packaged Sterile (Corning Cat# 357521 or equivalent)
  • Pipette Tips, Sterile, Filtered (P20, P200, P1000), any manufacturer
  • Wide-bore Pipette Tips, Sterile, Filtered (P200) (Mettler Toledo Cat# 30389241 or equivalent)
  • 1.5 mL DNA LoBind Tubes (Eppendorf Cat# 022431021)
  • 10 µL Disposable Inoculating Loops, Sterile, any manufacturer
  • Qubit Assay Tubes Thermo Fisher Scientific Cat# Q33252)

Equipment
  • Serological Pipette Controller, any manufacturer
  • Micropipettes (P20, P200, P1000), any manufacturer
  • Microcentrifuge (Eppendorf Cat# 5406000240 or equivalent)
  • Vortex (Benchmark Scientific Cat# BV101-B or equivalent)
  • 1x required, 2x recommended Thermo Mixer (Eppendorf Cat# 2231001127 or equivalent) containing 1.5 mL tube block
  • recommended Vertical Rotating Mixer (Thermo Fisher Scientific Cat# 15920D or equivalent)
  • Qubit Fluorometer (4.0, 3.0, or 2.0) (Thermo Fisher Scientific Cat# Q33238)

Safety warnings

Safety information
Chemical Safety Warning: Take proper precautions and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards.

NEB Monarch® HMW DNA Extraction Kit for Tissue: See NEB SDSs for additional information. Take proper precautions and wear appropriate PPE when handling reagents.
Proteinase K: GHS Category 1 for respiratory sensitization

Before start
  • Review the complete protocol before beginning.
  • Clean work surface and outer surface of pipettes with 70% ethanol before use.
  • Samples should be grown in broth overnight (grown from a single colony if possible).
  • Preheat thermal mixer with 1.5 ml block to 37°C (if available, preheat another to 56°C).
  • Chill 1x PBS.
  • Add ethanol (≥ 95%) to the gDNA Wash Buffer as indicated on the bottle label. Mark the bottle to indicate the date ethanol was added.

The volumes indicated in the protocol vary depending on input amounts. Refer to the following designations throughout the protocol to determine the appropriate volumes

PART 1: BACTERIAL LYSIS
4h
Pellet bacterial cells in a Monarch Pestle Tube by centrifugation at 16000 x g for 00:01:00 . If no Monarch Pestle Tubes are available, 1.5 ml Eppendorf tubes will suffice.
  • For E. coli: 1 x 109– 5 x 109; OR
  • Use about 1 ml of overnight culture per sample, or enough cells scraped from a plate to produce a similar sized pellet if cultured broth is not available or a viable option.
  • Increase starting amount of sample if DNA yield is insufficient; decrease if lysate remains cloudy.
1m
If not to be used on the same day, pelleted samples may be stored at -20 °C for up to a month. Avoid rethawing and refreezing samples; thaw frozen samples only once.
Prepare lysozyme.
Chicken lysozyme (25 mg/mL ) may be used for gram negative bacterial samples. Suspend 0.005 g in 200 µL of nuclease-free water for up to 20 samples.
For gram positive bacterial samples, MetaPolyzyme Multilytic Enzyme Mix may be used instead.
  • Resuspend a new tube of MetaPolyzyme Multilytic Enzyme Mix with PBS buffer (pH 7.5) and divide into aliquots of 200 µL (5 aliquots total per resuspended tube).
  • Tubes of suspended enzyme mix may be stored at -20 °C for future use.
Gram-negative and gram-positive bacteria are processed differently for the initial lysis steps.
Gram-negative Bacteria:
  • Resuspend pellet in 300 µL (Low Input: 150 µL ) cold PBS. Cold TE or Tris buffer may be used in place of PBS if preferred.
  • Add 10 µL of prepared Lysozyme and mix by vortexing briefly.
  • Add 300 µL (Low Input: 150 µl) HMW gDNA Tissue Lysis Buffer to the sample and mix by inverting 5-10 times.
  • Incubate at 37 °C in a thermal mixer with agitation at 1000 rpm for at least 02:00:00 .

The speed of the thermal mixer influences fragment length and lysis time. For most applications, maximum agitation speed (1400–2000 rpm) is recommended. For maximum gDNA size, agitate at 500 rpm. Incubation is complete when lysate turns clear, although in some cases it may be acceptable to proceed with a cloudy lysate (particularly samples of Salmonella and E.coli). Do keep in mind that incubation times may increase as agitation speeds decrease.
2h
Gram-positive Bacteria
  • Resuspend pellet in 150 µL cold PBS.
  • Add 10 µL of prepared MetaPolyzyme Multilytic Enzyme Mix and mix by inverting 5-10 times.
  • Incubate at 37 °C with no agitation for at least 02:00:00 (preferably 24 hours, if possible).
  • Add an additional 150 µL of cold PBS to the sample and mix by inverting 5-10 times.
  • Add 300 µL (Low Input: 150 µL ) HMW gDNA Tissue Lysis Buffer to the sample and mix by inverting 5–10 times.
2h
Lysed bacterial samples may be kept at 4 °C overnight (e.g., if samples are lysed on Monday, they can be stored in the fridge for use on Tuesday).
Following lysozyme treatment at 37°C, increase the temperature of the block in the thermal mixer to 56 °C .
Add 20 µL (Low Input: 10 µL ) of Proteinase K and mix by inverting 10–20 times.
Incubate at 56 °C for 00:30:00 in a thermal mixer at 1000 rpm .

The speed of the thermal mixer influences fragment length and lysis; higher agitation speeds reduce DNA size and sample lysis time. For most applications, including the standard ligation-based Oxford Nanopore Technologies (ONT) sequencing protocols, maximum agitation speed (1400-2000 rpm) is recommended to produce DNA fragments predominantly 50–250 kb. To achieve maximum gDNA size, up to the Mb range, use a low agitation speed. If sample remains cloudy after Proteinase K treatment, lysis has not worked properly and there will be no DNA yield.
30m
Add 10 µL (Low Input: 5 µL ) of RNase A and mix by inverting 5–10 times. Incubate for 00:10:00 at 56 °C with agitation in a thermal mixer at the speed used in Step 6.
10m
Add 300 µL (Low Input: 150 µL ) of Protein Separation Solution. Mix by inverting for 00:01:00 . Alternatively, a vertical rotating mixer at 20 rpm can be used.
1m
Centrifuge for 00:10:00 at 16000 x g . If working with multiple samples, during centrifugation, prepare the plastics for Part 2, as indicated in the following step.
  • The sample will separate into a large, clear upper phase (DNA) and a lower, clear phase (protein, usually on the bottom of the tube, but occasionally floating).
  • There may also be a white precipitate at the bottom of the tube.
  • Additional centrifugation time (10-20 minutes) may be required for complete phase separation, particularly when low agitation speeds were used.
10m
If working with multiple samples, prepare and label the plastics for the upcoming steps. Each sample will require:
  • Monarch Collection Tube II (no need to label)
  • One Monarch Bead Retainer inserted into the collection tube; this will be used to remove the wash buffer from the gDNA bound to the beads.
  • Two Monarch 2 ml Tubes; one for phase separation and one for elution.
  • One 1.5 ml microfuge tube (DNA low bind recommended, not provided); this will be used to collect the eluate.
Using a 200 µl wide-bore pipette tip, transfer the upper phase containing the DNA (large, clear phase) to a labeled Monarch 2 ml Tube.

A substantial fraction of HMW DNA will be located at the interface between the clear upper phase and the protein phase; highest yields will be achieved by transferring as much of the upper phase as possible. Avoid transferring material from the protein layer, though a small amount (1–2 µl) will not be detrimental. If protein enters the pipette tip, gently push it back into the tube. If a lower protein phase is not visible, leave ~30 µl behind to ensure protein is not carried over. Typically, the transferred volume will be ~ 800 µl (Low Input: ~400 µl). If the volume of the sample is < 700 µl (Low Input: < 350 µl), adjust the volume of isopropanol used in Step 2 of Part 2: HMW gDNA Binding and Elution to 0.7 volumes.
PART 2: HMW gDNA BINDING AND ELUTION
3h
Prepare an aliquot of isopropanol (550 µL per sample, with about 1 mL of overage).
Using a clean inoculating loop, add 2 DNA Capture Beads to each sample, which should be contained in a Monarch 2 ml Tube.
  • A single DNA Capture Bead should sit comfortably in the loop when the loop is held flat.
  • Flip the loop upside down to release the bead.
  • The same loop can be used for all tubes if it has not made contact with any of the sample tubes.
  • Discard loop after use.
Add 550 µL (Low Input: 275 µL ) or an adjusted amount of isopropanol (0.7 volume), close the cap, and invert slowly and gently by hand for 00:05:00 at the minimum . A manual inversion is complete when the tube returns to the upright position. Slow inversion is critical for the DNA to bind to the beads; each full inversion should take ~5–6 seconds. If necessary, flick the tube to release any beads that stick to the bottom of the tube.

After a 2–3 inversions, the solution becomes more viscous and the DNA will wrap loosely around the beads. During the following inversions, precipitation of gDNA may be visible, especially with larger input samples. The DNA complex will often contain small air bubbles. With increasing number of inversions, the DNA will completely wrap around the beads, often causing the beads to stick together. DNA binding to the beads should be complete after 25–30 inversions, and the solution should no longer be viscous. Additional inversions may be necessary for larger input samples.
5m
Remove and discard liquid by pipetting a p1000 pipette. Avoid removing any of the gDNA wrapped around the glass beads.
  • Angle tube so that beads remain at the bottom, and liquid reaches toward tube opening. Pipette from the liquid surface and continue to angle as liquid is removed (tube will be almost horizontal at the end)
  • For optimal DNA solubility, avoid letting the bound DNA dry out on the beads during this and the following steps; add the next buffer quickly.


Add 500 µL gDNA Wash Buffer, close the cap, and mix by inverting the tube 2–3 times. Remove the wash buffer as described in Step 15.
  • The loose gDNA complex will condense around the beads more tightly.
Repeat the wash in Step 16 and remove the wash buffer by pipetting. It is not necessary to remove all the gDNA Wash Buffer at this point.
Place a labeled Bead Retainer into a Monarch Collection Tube II. Pour the beads into the Bead Retainer and close the cap. Discard the used Monarch 2 ml Tube.
  • When working with multiple samples, be sure to close the cap of the Bead Retainer after each transfer of beads.


Pulse spin (≤ 1 second) the sample in a benchtop minicentrifuge to remove any residual wash buffer from the beads.
Separate the Bead Retainer from the collection tube, pour the beads into a new, labeled Monarch 2 ml Tube, and insert the used Bead Retainer into the labeled 1.5 ml microfuge tube for later use during elution. Discard the used collection tube.


Immediately add 100 µL Elution Buffer II onto the glass beads and incubate for a minimum of 00:05:00 at 56 °C in a thermal mixer with agitation at 300 rpm .
  • Halfway through the incubation, ensure the beads are not stuck to the bottom of the tube by tilting the tube almost horizontally and gently shaking. This ensures that the beads can move freely, allowing for optimal release of the DNA from the beads. It also ensures that the lower bead does not stick to the bottom of the tube during the following transfer step.
  • Elution volume can be reduced to as low as 50 µL without affecting recovery.
  • However, if using < 100 µl, the gentle shaking of the sample should be done several times during the incubation to ensure complete wetting of the beads.


5m
Ensure the Bead Retainer is inserted into the 1.5 ml microfuge tube (DNA low bind, not included). Pour the eluate and the glass beads into the Bead Retainer and close the cap.
  • When working with more than 1 sample, it is important to close the cap after each transfer of beads.
  • Typically, all the eluate flows into the bead retainer upon pouring. If any volume remains in the 2 ml tube, spin briefly and transfer.


Centrifuge for 00:00:30 at 12000 x g to separate the eluate from the glass beads. Discard the beads and retainer.
30s
Ensure that DNA is homogenously dissolved by pipetting and incubating.
Pipette eluate up and down 5–10 times with a wide-bore pipette tip and ensure any visible DNA aggregates are dispersed.
After pipetting, incubate at 37 °C for 01:00:00 , Overnight at Room temperature , or for longer at 4 °C .
2h
Samples can now be stored at 4 °C for future use.
  • Pipette up and down 5-10 times again before analyzing or using the HMW DNA.
  • When measuring the DNA concentration, use 2 µL of DNA and the Qubit™ dsDNA BR Assay Kit for higher accuracy.
Protocol references