Protocol for UHMW DNA Cleanup in the Oxford Nanopore Technologies® UL Library Prep Workflow
Prior to cleanup, prepare the following solutions:
- UL Cleanup Binding Buffer: 10 mM hexamine cobalt chloride1*, 10 mM Tris-HCl pH 8.0.
- UL Cleanup Wash Buffer: Use LFB Buffer (ONT #SQK-ULK001) or prepare 10% PEG 8000, 500 mM NaCl, 10 mM Tris-HCl pH 8.0
- Elution Buffer: Use EB Buffer (ONT #SQK-ULK001) or 10 mM Tris-HCl pH 8.0
*Alternatively, Oxford Nanopore Technologies recommends 16 mM spermine in place of hexamine cobalt chloride. Both compounds demonstrate similar precipitation and binding properties.
Other Materials Required:
- Monarch DNA Capture Beads (NEB #T3005L)
- Monarch 2ml Tubes (NEB #T3003L)
- Monarch Bead Retainers (NEB #T3004L)
- DNA low bind tubes
- Oxford Nanopore Technologies Ultra-Long DNA Sequencing Kit (SQK-ULK001)
UHMW DNA Cleanup After Tagmentation & Rapid Adapter Addition:
Follow Ultra-Long DNA Sequencing Kit (SQK-ULK001) Protocol. Logging into the Nanopore Community may be required to access this protocol.
- Post incubation with RAP F Adapter, transfer the library to a Monarch 2 ml Tube (NEB #T3003) by pouring carefully or pipetting with a wide-bore pipette tip.
- Add 2 Monarch DNA Capture Beads (NEB #T3005) to the library.
- Add 1 volume of the UL Cleanup Binding Buffer, and slowly invert manually until DNA has bound to the beads and no further compacting is observed (up to a maximum of 25 times). A non-alcohol-based binding buffer is used for optimal performance of the motor protein on the adapter. DNA binding with this formulation is faster than when isopropanol is used in the standard extraction workflow, and often 15 inversions are sufficient. If the DNA is not wrapping tightly around the beads, hold the tube with the cap down at a 45-degree angle and roll it 5-10 times around its center to compact the precipitate more tightly around the beads.
- Remove supernatant by pipetting. Gently push beads to the side for complete removal of the liquid.
- Add 1 ml UL Cleanup Wash Buffer and invert 2-3 times. Incubate 3 minutes at room temperature, then remove buffer by pipetting as described above.
- Repeat the wash step.
- Pulse spin very briefly (<1 second) in a benchtop minifuge to collect drops at the bottom of the tube. Do not let the minifuge reach full speed. Carefully remove traces of wash buffer from under the beads by pipetting with a fine pipette tip. A few µl of wash buffer may remain on the beads as dead volume, but this will not affect the downstream performance.
- Add 225 µl Elution Buffer (see above) for 40 µg DNA samples (PromethION) or 115 µl Elution Buffer for 20 µg samples (MinION). If quantitation of the library is planned2, increase the elution volume with 10 µl to 235 µl and 125 µl, respectively.
- Incubate for 30 minutes at 37°C. During incubation gently pipette and dispense the eluate over the glass beads using a wide bore pipette tip to aid elution. Repeat if necessary; solution should show increased viscosity caused by release of the DNA from the beads.
- Place a Monarch Bead Retainer in a 1.5 ml DNA low bind tube. Pour eluate with beads into the bead retainer and spin for 1 minute at maximum speed (>16,000 x g). Confirm that DNA was completely released from the beads. If after the spin, DNA threads are visible between beads and eluate, centrifuge again for 1 minute at maximal speed (>16,000 x g).
- Discard bead retainer with beads and close tube with eluate.
UHMW DNA Library Resuspension:
Carefully and slowly pipette eluate up and down 5-10 times using a P200 wide-bore tip to resuspend the DNA complex.
- Incubate samples for 5-10 minutes at room temperature.
- Repeat steps 1-2 until sample has become a homogenous viscous liquid. This may take between 30 and 90 minutes. Libraries can also be stored at 4°C for further homogenization. The sample is now ready for the flow cell loading process. Optional: confirm DNA concentration2.
- Cahyani, I., Tyson, J., Holmes, N., Quick, J., Loman, N., Loose, M. “FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms” Protocols.io, (09/2021) https://dx.doi.org/10.17504/protocols.io.bxwrppd6
- Koetsier PA, Cantor EJ. A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA. BioTechniques 71 (2), 439-444 (2021). https://doi.org/10.2144/btn-2021-0051
- ’ Ultra-Long (UL) DNA Sequencing®Protocol Guidance for Extraction of UHMW DNA using the Monarch HMW DNA Extraction Kit Upstream of Oxford Nanopore Technologies
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Measuring, Analyzing & Storing High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits
- Monarch HMW DNA Extraction Kit for Cells & Blood Product Manual
- Monarch HMW DNA Extraction Kit for Tissue Product Manual