Sep 26, 2025

Public workspaceSanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.5

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1128ovr2/v1
Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.5
  • Graeme Oatley1,
  • Amy Denton1,
  • Anna Kovalevskaia2,
  • Caroline Howard1
  • 1Tree of Life, Wellcome Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA;
  • 2Wellcome Sanger Institute
  • Tree of Life at the Wellcome Sanger Institute
  • Earth BioGenome Project
Amy Denton
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1128ovr2/v1
Protocol Citation: Graeme Oatley, Amy Denton, Anna Kovalevskaia, Caroline Howard 2025. Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.5. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1128ovr2/v1
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: September 04, 2025
Last Modified: September 26, 2025
Protocol Integer ID: 226421
Keywords: HMW DNA extraction, magnetic bead extraction, automated DNA extraction, MagAttract, Plant HWM DNA extraction, solid phase reversible immobilisation, reference genome, long read sequencing, sanger tree of life hmw dna extraction, automated extraction of hmw dna, qiagen magattract, hmw dna extraction kit, hmw dna fragmentation, hmw dna pooling, pacbio sequencing, automated plant magattract, automated extraction, plant species, tree of life programm, automated fungi extractions, KingFisher Apex, KingFisher, life hmw dna extraction, spri of hmw dna, qiagen magattract hmw dna extraction kit, recoveries of hmw dna, hmw dna, cryoprepped tissue samples from plant, yield of dna, tree of life programme, cryoprepped tissue sample, high molecular weight spri, acronyms hmw, genome size of the species, dna, fungi
Funders Acknowledgements:
Wellcome Trust
Grant ID: 206194
Wellcome Trust
Grant ID: 218328
Gordon and Betty Moore Foundation
Grant ID: GBMF8897
Abstract
This protocol is for the automated extraction and SPRI of HMW DNA from cryoprepped tissue samples from plants and fungi intended for long-read sequencing using the Qiagen MagAttract HMW DNA extraction kit and the Thermo Fisher KingFisher™ Apex. This process is effective for a wide variety of plant species covered by the Tree of Life Programme. The output of this protocol is HMW DNA, which – depending upon yield and genome size of the species – can be directed towards either HMW DNA Pooling, HMW DNA Fragmentation: Opentrons® OT-2 for PacBio LI, HMW DNA Fragmentation: Diagenode Megaruptor® 3 for LI PacBio or HMW DNA Fragmentation: Covaris g-Tube for ULI PacBio. This protocol was adapted from Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.4 to include an overnight elution in order to maximise the yield of DNA, as well as an updated pre-shear SPRI to improve recoveries of HMW DNA.

Acronyms
HMW: high molecular weight
SPRI: solid-phase reversible immobilisation
LI: low input
ULI: ultra-low input
Guidelines
  • For the lysis buffer master mix, prepare enough for n+1 samples to account for pipetting errors.
  • Keep samples on dry ice to maintain temperature and prevent nucleic acid degradation until the lysis buffer is ready to be added to them.
  • For the 0.45X SPRI, the DNA and AMPure beads should not sit together in the sample plate for more than 5 minutes before starting the KingFisher™ Apex.
  • An experienced operator can expect to comfortably process up to 32 samples, with approximately 2–3 hours handling time over a start to finish period of 4–5 hours. This estimation includes the utilisation of the KingFisher™ Apex for both the extraction and SPRI protocols, and excludes the overnight elution and subsequent QC checks.


Additional Notes:
  • FluidX tubes are used throughout the Tree of Life programme in order to track samples, therefore rather than the microcentrifuge tubes which have been mentioned in this protocol for DNA storage, all routine DNA extracts are stored in FluidX tubes.
  • Both the KingFisher™ Apex protocol scripts and the KFX.files have been made available for this protocol - the KFX.files require ‘BindIx software for KingFisher Apex’ to allow the KingFisher™ Apex protocols to be viewed on a PC or laptop. Alternatively, the files can be transferred directly onto a KingFisher™ Apex instrument using a USB flash drive.
Materials
  • 1.5 mL DNA Lo-Bind microcentrifuge tubes (Eppendorf Cat. no. 0030108051)
  • 2 mL DNA Lo-Bind microcentrifuge tubes (Eppendorf Cat. no. 0030108078)
  • Thermo Fisher KingFisher™ 96-well Deep-well plates (Thermo Fisher Cat. no. 95040450)
  • Thermo Fisher KingFisher™ 200 µL standard 96-well Plate (Thermo Fisher Cat. no. 97002084)
  • Thermo Fisher KingFisher™ 96 Tip Comb (Thermo Fisher Cat. no. 97002570)
  • Qiagen MagAttract HMW DNA extraction kit (Qiagen Cat. No. 67563)
  • Dry ice
  • 1 x phosphate-buffered saline (PBS)
  • 100% absolute ethanol
  • 15 mL or 50 mL centrifuge tubes
  • AMPure PB beads (Pacific Biosciences Cat. no. 100-265-900)
  • Buffer EB (Qiagen Cat. no. 19086)

Equipment:
  • Pipettes for 0.5–1000 μL and filtered tips
  • Wide-bore tips (200 μL and 1000 μL, filtered if available)
  • Thermo Fisher KingFisher™ Apex instrument (Cat. no. 5400930)
  • Corning® CoolRack (Cat. no. 432051) or equivalent
  • Eppendorf ThermoMixer C (Cat. no. 5382000031)
  • Eppendorf SmartBlock 2.0 mL (Cat. no. 5362000035)
  • Eppendorf™ Centrifuge 5425/5425 R (Cat. no. 5405000263)
  • Vortexer (Vortex Genie™ 2 SI-0266)
  • Mini centrifuge (Cat. no. SS-6050)
  • DynaMag™-2 magnetic rack (Cat. no. 12321D)
  • Timer

KingFisher™ Apex DNA Extraction Protocol Script:
KFX file: Download Qiagen MagAttract Overnight elution.kfxQiagen MagAttract Overnight elution.kfx2KB

  1. Pick Up Tip - Tip Plate
  2. DNA Binding - Sample Plate Pre-collect beads: Off Release beads: Off Heating & Cooling: Off Mixing 1# 00:05:00 Fast Postmix: Off Collect beads: On 5 Count 2 Seconds
  3. Collect Beads 1 - Sample Plate Collect beads: Count 5 Collect time 1 Second
  4. Wash 1 - MW1 Wash 1 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
  5. Collect Beads 2 - MW1 Wash 1 Plate Collect beads: Count 5 Collect time: 1 Second
  6. Wash 2 - MW1 Wash 2 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
  7. Collect Beads 3 - MW1 Wash 2 Plate Collect beads: Count 5 Collect time: 1 Second
  8. Wash 3 - PE Wash 1 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
  9. Collect Bead 4 - PE Wash 1 Plate Collect beads: Count 5 Collect time: 1 Second
  10. Wash 4 - PE Wash 2 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
  11. Collect Bead 5 - PE Wash 2 Plate Collect beads: Count 5 Collect time 1 Second
  12. Water Rinse - NFW Plate Pre-collect beads: Off Release beads: Off Heating & Cooling: Off Mixing 1# 00:00:00 Postmix: Off Collect beads: On 5 Count 1 Second
  13. Dry - NFW Plate Duration: 00:01:00 Dry Type: Above Well
  14. Elute - Elution Plate Pre-collect beads: Off Release beads: On 00:00:00 Heating & Cooling: On 25°C Pre-heat: Off Mixing 1# 00:00:05 Medium Looping: 1 2# 00:05:00 Slow Postmix: Off Collect beads: Off
  15. Leave Tip - Tip Plate

KingFisher™ Apex Bead Collection Protocol:

KFX file: Download Qiagen MagAttract Overnight elution_bead collection.kfxQiagen MagAttract Overnight elution_bead collection.kfx1KB

  1. Pick Up Tip - Tip Plate
  2. Elute - Elution Plate Pre-collect beads: Off Release beads: On 00:00:00 Heating & Cooling: On 25°C Pre-heat: On Mixing 1# 00:00:05 Medium Looping: 1 2# 00:02:00 Slow Postmix: On 00:00:00 Medium Collect beads: On 3 Count 1 Second
  3. Collect Beads - Elution Plate Collect beads: Count 5 Collect time: 1 Second
  4. Leave Tip - Tip Plate

KingFisher™ Apex 0.45X SPRI Protocol:


KFX file: Download 0.45X SPRI 30 min elution + mix.kfx0.45X SPRI 30 min elution + mix.kfx2KB


  1. Pick Up Tip - Tip Plate
  2. Mix - Sample Plate Pre-collect beads: Off Release beads: On 00:00:00 Heating & Cooling: Off Mixing: 1# 00:01:00 Slow 2# 00:01:00 Medium 3# 00:08:00 Paused Looping: 1 Tip position: Tip edge in liquid Postmix: Off Collect beads: On 10 Count 30 Seconds
  3. Wash 1 - Ethanol Wash Plate Pre-collect beads: On Release beads: Off Heating & Cooling: Off Mixing 1# 00:00:30 Slow Postmix: Off Collect beads: Off
  4. Wash 2 - Ethanol Wash Plate Pre-collect beads: Off Release beads: Off Heating & Cooling: Off Mixing 1# 00:00:30 Slow Postmix: Off Collect beads: Off
  5. Dry - Ethanol Wash Plate Duration: 00:01:00 Above well
  6. Elute - Elution Plate Pre-collect beads: Off Release beads: On 00:01:00 Slow Heating & Cooling: On 37℃ Preheat: On Mixing: 1# 00:00:50 Medium 2# 00:14:00 Slow 3# 00:00:10 Fast Looping: 2 Tip position: Tip edge in liquid Postmix: Off Collect beads: On 4 Count 30 Seconds
  7. Leave Tip - Ethanol Wash Plate


Protocol PDF: Download Sanger Tree of Life HMW DNA Extraction_ Automated Plant MagAttract v.5.pdfSanger Tree of Life HMW DNA Extraction_ Automated Plant MagAttract v.5.pdf208KB

Troubleshooting
Safety warnings
  • The operator must wear a lab coat, powder-free nitrile gloves and safety specs to perform the laboratory procedures in this protocol. Cotton glove liners are strongly recommended when handling the samples on dry ice.
  • Waste needs to be collected in a suitable container (e.g. plastic screw-top jar or Biobin) and disposed of in accordance with local regulations.
  • Liquid waste needs to be collected in a suitable container (e.g. glass screw-top jar) and disposed of in accordance with local regulations.
  • Do not open the door of the KingFisher™ Apex instrument whilst it is in operation.
Before start
  • Add 100% ethanol to the MW1 and PE wash buffers as per manufacturer’s instructions.
  • AMPure PB beads are stored in the fridge at 4 °C – take them out 30 minutes before starting the 0.45X SPRI KingFisher™ Apex protocol to bring them to room temperature.
Sample Lysis
Prepare a lysis buffer master mix in a 50 mL centrifuge tube:

ReagentVolume per sample
Phosphate-buffered saline (PBS)200 µL
Buffer AL150 µL

Place the lysis buffer on the 65 °C heat block and incubate at 400 rpm for at least 20 minutes. Keep at temperature until added to the sample.
Transfer 50 mg of cryogenically disrupted tissue from each sample to 2 mL microcentrifuge tubes.
Ensure the disrupted tissue is completely disrupted into a fine powder; avoid matted/clumped powder. This is crucial for optimal DNA yield and integrity; poorly disrupted tissue drastically decreases lysis and extraction efficiency.
Any samples containing poorly disrupted tissue ‘chunks’ should be further cryogenically disrupted.
Transfer the samples to a pre-chilled cold block on wet ice and incubate for 10 minutes to equilibrate temperature.
Add 20 µL Proteinase K (for n+1 samples) to the preheated lysis buffer immediately prior to initiating lysis, swirling the centrifuge tube to mix.
Add 370 µL of the preheated lysis buffer plus Proteinase K to each sample, immediately homogenising the lysate by mixing with 5 rapid pulse vortexes, and place on the 55 °C heat block at 600 rpm for 15 minutes.
After 5 minutes incubation, resuspend any severely aggregated samples by pipette mixing with a wide-bore pipette tip.
After the initial 15 minute incubation, add 4 µL RNase A to each sample and mix thoroughly by inversion until any aggregated, insoluble or sedimented tissue particles are resuspended.
Incubate samples for a further 45 minutes on the heat block at 55 °C at 600 rpm.
For the final 15 minutes of lysis, remove the 600 rpm mixing from the heat block to allow aggregated, insoluble or sedimented tissue particles to settle at the bottom of the tube - this will reduce gDNA loss during later centrifugation.
Loading and Running the KingFisher™ Apex for DNA Extraction
Whilst samples are lysing, label nine 1 mL 96-well deep-well KingFisher™ plates and fill the number of wells required for the number of samples in each plate as follows:

PlateReagent(s) required
Tip plate96-well tip comb (no reagent)
Elution plate 410 µL Buffer AE
NFW Wash 500 µL Nuclease-Free Water
PE Wash 2700 µL Buffer PE
PE Wash 1700 µL Buffer PE
MW1 Wash 2700 µL Buffer MW1
MW1 Wash 1700 µL Buffer MW1
Sample plate40 µL Suspension G magnetic beads + 280 µL Buffer MB

Once samples have completed lysing, remove sample tubes from the heat block and allow the lysate to settle to the bottom of the tube for 5 minutes.
Centrifuge the samples at 8000 rpm for 10 minutes at room temperature.
Using a wide-bore pipette tip, set the volume to 380 µL, transfer lysate from the sample tubes to individual wells in the sample plate, taking care not to transfer large pieces of debris if possible.
Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details in the KingFisher™ Apex DNA Extraction Protocol section/attached file within Materials) and select using the play button.
Load the filled plates onto the instrument following the instructions provided on screen.
Once the final plate is loaded, the protocol will automatically begin; this takes approximately 40 minutes.
Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument. Make sure to retain the Tip Plate which contains the tip comb for future steps.
Seal the Elution Plate with a plate seal and leave overnight at room temperature to allow the DNA to elute.
The following morning, remove the plate seal and return to the KingFisher™ Apex in order to run the ‘bead collection’ protocol. Select from the protocol list and load the Tip Plate and Elution Plate as prompted by the instrument.
Once the bead collection protocol has completed after approximately 5 minutes, follow the on-screen instructions to remove the plates from the instrument.
Inspect the Elution Plate for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the DNA using a wide bore pipette tip. Return samples back to their original well following bead removal.
The Elution Plate is now the ‘Sample Plate’ for the 0.45X SPRI.
Loading and Running the KingFisher™ Apex for the 0.45X SPRI
Set-up the KingFisher™ plates for the 0.45X SPRI as detailed below:

PlatePlate TypeReagent(s) required
Tip Plate1 mL deep-well96-well tip comb (no reagent)
Sample Plate (Elution Plate 1 from DNA Extraction Protocol)1 mL deep-well380 µL DNA + 171 µL AMPure PB beads
Ethanol Wash Plate1 mL deep-well1000 µL 80% EtOH (freshly made)
Elution Plate200 µL standard135 µL Buffer EB

Select the required 0.45X SPRI protocol in the protocol list on the KingFisher™ Apex (details in the KingFisher™ Apex 0.45X SPRI Protocol section/attached file within Materials) and select using the play button.
Load the filled plates onto the instrument, following the instructions provided on screen.
Once the final plate is loaded, the protocol will automatically begin; this will take approximately 50 minutes.
Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.
Using a wide-bore pipette tip, transfer the 130 µL of eluate from the elution plate into microcentrifuge tubes.
Perform the required QC to determine the quantity and quality of the DNA extracted.
Store the DNA at 4 ºC.
Protocol references