May 12, 2026
Version 2
FLEX(-) Bacterial DNA Extraction Protocol for Nanopore and Hybrid Whole Genomic Sequencing V.2
- *Jack Boulé1,2,
- *Karan Desai1,2,
- Robert Rebelo1,
- Evelyn Wisebourt1,
- Mohamad Ali1,
- Golnoush Akhtari1,
- Tony Mazzulli1,2,3,
- Susan M Poutanen1,2,3
- 1UHN/Mount Sinai Department of Microbiology, Toronto;
- 2UofT Department of Laboratory Medicine and Pathobiology;
- 3Department of Medicine, University of Toronto, Toronto, Ontario, Canada
- *Jack Boulé: *These authors contributed equally;
- Mount Sinai Microbiology
Protocol Citation: *Jack Boulé, *Karan Desai, Robert Rebelo, Evelyn Wisebourt, Mohamad Ali, Golnoush Akhtari, Tony Mazzulli, Susan M Poutanen 2026. FLEX(-) Bacterial DNA Extraction Protocol for Nanopore and Hybrid Whole Genomic Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3n67zv25/v2Version created by Jack Boulé
Manuscript citation:
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: May 12, 2026
Last Modified: May 12, 2026
Protocol Integer ID: 316890
Keywords: Nanopore, DNA Extraction, Extraction, Eluate, Long fragment, rapid extraction, fast, Sequencing, Illumina, dna extraction protocol for nanopore sequencing, nanopore sequencing, dna extraction protocol, nanopore, department of clinical microbiology, clinical microbiology, fragment genomic dna from gram, suitable for downstream molecular workflow, viridans group streptococci, enterococcus, read sequencing, positive bacteria, fragment genomic dna, downstream molecular workflow, hemolytic streptococcus, staphylococcus, bacterial dna extraction protocol for nanopore, bacterial dna extraction protocol, using oxford nanopore r10 chemistry, oxford nanopore r10 chemistry, oxford nanopore, dna extraction, addition to nanopore, bacterial dna, extracted dna, genome sequencing, fragment length for downstream molecular application, downstream molecular application, sequencing, specific extraction option, single extraction, bacteria, duplicate extraction step, dna, dna with sufficient purity, extraction, avoiding duplicate, Long-read, long read, long read, bacterial dna
Disclaimer
Please note: although this workflow may be broadly applicable to Gram-negative bacteria, it has been validated only for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter spp., Stenotrophomonas maltophilia, and Serratia marcescens.
Abstract
This protocol provides a rapid, high-throughput, and cost-effective method to extract high-quality, long-fragment genomic DNA from Gram-positive bacteria (e.g., Staphylococcus, Enterococcus, β-hemolytic Streptococcus, and viridans group streptococci). The workflow is optimized to produce DNA with sufficient purity, concentration, and fragment length for downstream molecular applications, with a primary focus on whole-genome sequencing using Oxford Nanopore R10 chemistry (including library preparation requirements).
In addition to Nanopore sequencing, the extracted DNA is suitable for common downstream workflows such as qPCR and other library preparation applications. The protocol can also be used for Illumina short-read sequencing; however, faster Illumina-specific extraction options may be available. In settings where both Nanopore and Illumina sequencing are performed in parallel, this protocol enables a single extraction to support both workflows, avoiding duplicate extraction steps.
This method has been validated by the Department of Clinical Microbiology at Mount Sinai Hospital, Toronto, and is currently being being used in clinical validation clinical validationworkflows.
Guidelines
Definitions and Abbreviations:
- FLEX(+): Fast, Long-fragment, EXtraction for Gram + bacteria.
- SPRI: Solid-Phase Reversible Immobilization paramagnetic bead chemistry.
- RT: Room temperature (18–25 °C).
Materials
Reagents
- TE buffer (pH 8.00, filtered): 10 mM Tris, 1 mM EDTA
- Sarkosyl (N-lauroylsarcosine; stock): 10% (prepared in ddH₂O or TE buffer)
- Lysozyme (stock): 100 mg/mL (prepared in ddH₂O or TE buffer)
- Proteinase K (stock): 20 mg/mL (prepared in ddH₂O or TE buffer)
- SPRI-Select paramagnetic beads: Beckman Coulter
- Molecular-grade water: ddH₂O
- Ethanol (fresh): 80% (v/v) for bead washes
Consumables
- 0.5 mm glass bead tubes: Qiagen 0.5mm Glass PowerBead tubes with screw caps (or equivalent)
- Inoculation loops: sterile 1 µL loop (or 10 µL loop)
Collection vessels (choose one):
- 1.5 mL low-bind microcentrifuge tubes, or
- 0.8 mL “midi” or PCR 96-well plate
- Pipette tips: aerosol-resistant filter tips (20–1000 µL range)
Equipment
- Vortex mixer: compatible with a Qiagen PowerBead vortex adapter top
- Qiagen PowerBead vortex adapter top
- Heating: calibrated temperature blocks or water baths at 37 °C and 56 °C
- Magnetic separation (choose one):
- Thermo DynaMag rack (for 1.5 mL tubes), or
- Illumina-style 96-well magnetic rack (for midi plates)
- DNA quantification: Qubit fluorometer (HS dsDNA assay) and NanoDrop (or equivalent spectrophotometer)
- Fragment analysis (optional/as available): TapeStation, Femto Pulse, or equivalent system
Troubleshooting
Problem
If concentration QC fails: Too low
Solution
Discard the eluate and rerun the protocol with more biomass than initial run.
Problem
If concentration QC fails: Too high
Solution
Simply dilute the final eluate with more ddH2O until appropriate
Problem
If contamination QC fails:
Solution
Perform another SPRI Cleanup on the final eluate, but use 100 µL of SPRI beads instead of original 225 µL.
Problem
If fragment analyses QC fails: Too short
Solution
Discard eluate, repeat full FLEX protocol with 3 min of bead beating instead of 5 min.
Problem
If fragment analyses QC fails: Too long
Solution
Discard eluate, repeat full FLEX protocol with 8 min of bead beating instead of 5 min.
Safety warnings
Throughout the protocol, particularly the section that utilizes magnets, it is recommended that you review the actual mechanism behind DNA binding, and understand the chemistry of the SPRI select beads. This makes troubleshooting, as well as appropriate removal from the magnet, much more intuitive.
Warning: Sarkosyl (N-lauroylsarcosine) is an irritant and can be harmful if inhaled or if it contacts skin/eyes—handle it in a fume hood when possible, wear appropriate PPE (lab coat, nitrile gloves, eye protection), avoid generating aerosols, and clean spills immediately according to your institution’s chemical safety procedures.
Before start
Specimen Collection
- Specimen: Single pure isolate from a confluent purity plate (18–24 h typical, organism-appropriate conditions).
- Transport/Storage: Room temperature (RT) bench-top during setup
- Rejection criteria: Mixed culture, plate age is over 72 h (discretionary), or visible contamination on the media.
- Recommendations:
- Confirm species ID using MALDI-TOF or other equivalent identification mechanism.
- Use an ethanol safe marker/pen when labelling tubes to prevent washing of labels
FLEX(-) Extraction Preparation
Label a 1:1 matched set of PowerBead tubes and LoBind tubes for each isolate accession (use the same sample ID format across all tubes). Then label the collection vessels using the same IDs—either:
- 1.5 mL Eppendorf tubes (recommended for ≤48 samples), or
- a 0.8 mL 96-well PCR plate (recommended for >48 samples).
- Note: Each option requires a different magnet type (96-well plate magnet if using a PCR plate, and a standing tube magnet for Eppendorfs (such as the Thermofisher DynaMag)); ensure you have the appropriate magnet.
Pre-equilibrate a temperature block or water bath to 56 °C (either may be used based on personal preference/equipment availability).
Attach the Qiagen Vortex Adapter top to a Vortex-Genie (or equivalent vortex mixer).
Note: The adapter top holds up to 24 samples; use multiple adapter tops in parallel if processing more than 24 samples at once.
Calculate how much of each enzyme is required for given batch size (See Table 1).
Table 1:
| A | B | |
| Reagent: | Proteinase K 20mg/mL | |
| Volume/Sample: | 5µL |
Calculate the amount of each reagent required for a given batch size (See Table 2).
Table 2:
| A | B | C | D | E | F | |
| Reagent: | TE Buffer (10 mM Tris, 1 mM EDTA), pH 8.00) | SPRI Select Beads (Beckman Coulter) | Fresh 80% Ethanol | Molecular Grade Water (ddH2O) | 10% Sarkosyl Solution | |
| Volume/Sample: | 300µL | 225µL | 1200µL | 100µL | 8µL |
Remove the enzyme (Proteinase K) from the freezer and allow it to thaw. Keep at 4 °C until ready for use.
Aliquot 300 µL of TE Buffer into each 0.5mm Glass Powerbead Tube.
Inoculate each PowerBead tube with isolated bacterial colonies using either a heaping 1 µL loop or half of a 10 µL loop, while minimizing agar carryover.
Note: After inoculation, gently spin/rotate the loop in the tube to help disperse and homogenize the biomass before proceeding.
Membrane Solubilization
Pipette 8μL Sarkosyl (10% sol), (Using a P10) into each Powerbead tube, directly into the solution
Slowly and gently pipette mix up and down 2-3 times.
Over-pipetting may lead to foam that makes later steps far more difficult. In the case this occurs, allow foam to settle, gently tapping the tube to speed up the process. Some foam is expected.
Mechanical Lysis
Insert Powerbead tubes into a Qiagen Vortex Adapter Top, with the cap side facing inward.
Place the top onto a Vortex Genie or an equivalent vortex.
Set the vortex speed to 9 (or the max speed on an equivalent Vortex), and allow the top to spin for 5 minutes.
Note: If using an alternative Vortex, samples may spin faster than expected, causing over fragmentation of DNA, this can be corrected for by lowering the speed until fragment analysis returns optimal results)
Turn off the vortex and remove the adapter top.
Place the tubes back onto a rack, maintaining the original format.
Denaturation of Interfering Proteins
Pipette 5 µL of Proteinase K (20 mg/ml) (Using a P10) into each Powerbead tube, directly into solution.
Slowly and gently pipette mix up and down 2-3 times.
Place Powerbead tube into a 56 °C dry incubator or a 56 °C water bath, maintaining the original layout of the tubes on their respective racks.
Incubate for 15 min.
Remove from incubator.
Removal of Contaminants/DNA Binding
Transfer 225 µL of the solution from Powerbead tubes into either a 0.8 ml, 96 well MIDI/PCR Plate, or a 1.5 ml Eppendorf tube (see note in step 1)
Note: You may find that the powerbeads are sucked up into the pipette. This can be solved by tilting the tube sideways and drawing solution from the side beads are not sitting on.
Add 225 µL of SPRI Select Beads into each well/tube.
Note: a 1:1 ratio of SPRI select beads to solution ensures you select for fragments of all sizes. In the case you changed any of the initial volumes used, adjust SPRI bead volume accordingly.
Mix by pipetting up and down 3-5 times.
- Homogeneity is critical for this step as too little mixing will prevent complete DNA binding.
Place onto appropriate magnet (96 well Illumina Magnet for MIDI plates, Thermo DYNAMag Magnet for Eppendorfs).
Leave on magnet until lysate appears clear (3-5 min), and a pellet has formed on the well/tube side wall.
Note: If using eppendorfs, rotating the tube left and right while on the magnet can speed up the process of pellet formation.
With the plate/tube STILL on the magnet, carefully remove the supernatant with a P1000 set to 1000 µL. Avoid touching the pellet. If any pellet is sucked into the pipette, put it back into the well/tube, towards the magnet side, and leave for another 2 minutes before removal of supernatant.
It is highly recommended that you review the mechanism behind SPRI select beads to best understand and proactively troubleshoot proper lysate removal.
Ethanol Washes x 2
With the following steps, you must put on and take off the plate/tube from the magnet as stated. Failure to do so will result in the complete loss of eluate.
Remove the tube/plate from the magnet.
Pipette 600 µL of 80% ethanol into each well/tube directly onto the pellet.
Resuspend the pellet in the ethanol by pipetting up and down until homogeneous.
Note: Some pellets may require scraping with the tip of the pipette to help them better homogenize.
Place tube/plate back onto the magnet.
Allow pellet to reform (2-3 min).
Remove supernatant with a P1000 set to 1000 µL.
Remove tube/plate from magnet again.
Pipette 600 µL of 80% ethanol into each well/tube directly onto the pellet.
This time, do not resuspend the pellet fully by mixing, simply allow the ethanol to sit on the pellet.
Place the tube/plate back onto the magnet.
Wait 2-3 min.
Remove and discard the supernatant with a P1000 set to 1000 µL, carefully avoiding the pellet.
Allow pellet to air dry for 10 min, monitoring and wicking away any residual pooling; do not over-dry to the point of visible cracking. That said, it is better to over-dry than under-dry, as any remaining ethanol will act as a contaminant in your final eluate.
Once dry, take the tube/plate off the magnet.
Final DNA Elution
Add 100 µL of Molecular Grade Water (ddH2O) directly onto the pellet.
Note: if you are finding that your yield is too low using 100µL, you may decrease the volume accordingly.
Resuspend pellet fully, by mixing up and down with a pipette.
Place tube/plate back onto the magnet.
Wait 2-3 min for pellet to reform.
Transfer all 100 µL of the supernatant into a labelled Lo-Bind Eppendorf tube, this is your final DNA solution, which can now be stored at -80 °C for up to 10 years.
You may now discard the tubes/plate with the pellet (DNA is now in the supernatant you just realiquoted)
Eluate Quality Control (Optional, but highly recommended)
Quantify DNA with a Qubit Fluorometer, using an eluate input of 4 µL, and a High Sensitivity Reagent input of 196 µL. (See Oxford Nanopore Technologies NO-MISS Protocol for acceptable eluate concentrations)
Check for contamination using a Nanodrop or equivalent device. (See Oxford Nanopore Technologies NO-MISS Protocol for acceptable 260/280 and 260/230 ratios)
Optionally: Check for high fragment length retention using a Bioanalyzer or Fragment Analyzer (See Oxford Nanopore Technologies NO-MISS Protocol for acceptable fragment lengths)
See Troubleshooting Guide for related fixes
Acknowledgements
This research was supported by the Lunenfeld-Tanenbaum Research Institute (LTRI) at Sinai Health. We thank Kin Chan and associates for their guidance and resources.