- Materials
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- Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
- Monarch® HMW DNA Extraction Kit for Tissue (New England Biolabs, T3060)
- Flow Cell Wash Kit (EXP-WSH004)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- 15 ml Falcon tubes
- Isopropanol, 100% (Fisher, 10723124)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Thermal cycler or heat block
- Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
- Vortex mixer
- Microfuge
- Wide-bore pipette tips
- P1000 pipette and tips
- P200 pipette and tips
- Qubit fluorometer (or equivalent)
- Ice bucket with ice
- Timer
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For this protocol, you will need to extract gDNA from 6 million cells in 40 µl PBS before starting the library preparation.
This protocol has been developed using the NEB Monarch® HMW DNA Extraction Kit for Tissue (cat # T3060). Alternative kits are available from NEB which are specifically designed for the extraction from blood and cells. However, they have not been validated by Oxford Nanopore Technologies.
This method has been validated for use on the following inputs:
- 6 million white blood cells isolated from 1.6 ml blood (bovine), using RBC Lysis Solution (QIAGEN, cat # 158904)
- 6 million cells isolated from 1 g frozen tissue, using pluriSelect Cell Straining equipment.
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Ultra-Long DNA Sequencing Kit (SQK-ULK114) contents
Name Acronym Cap colour Number of vials Fill volume per vial (µl) Rapid Adapter RA Green 1 40 Fragmentation Mix FRA Amber 1 50 FRA Dilution Buffer FDB Clear 1 1,600 Elution Buffer EB Black 2 1,500 Extraction EB EEB Orange 3 1,700 Sequencing Buffer UL SBU Red 2 1,000 Loading Solution UL LSU White cap, pink label 1 200 Flush Tether UL FTU Purple 1 600 Flow Cell Flush FCF Blue 2 15,500 Precipitation Buffer PTB Blue 2 1,700 Precipitation Star PS Yellow 6 1 star -
Flow Cell Wash Kit (EXP-WSH004) contents
Contents Volume (µl) No. of tubes No. of uses Wash Mix (WMX) 15 1 6 Wash Diluent (DIL) 1,300 2 6 Storage Buffer (S) 1,600 2 6 - Wash Mix (WMX) contains DNase I.
- Wash Diluent (DIL) contains the exonuclease buffer that maximises activity of the DNase I.
- The Storage Buffer allows flow cells to be stored for extended periods of time.
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To maximise the use of the Ultra-Long DNA Sequencing Kit V14, the EEB Expansion (EXP-EEB001) and the Ultra-Long Auxiliary Vials (EXP-ULA001) expansion packs are available.
These expansions provide extra library preparation and flow cell priming reagents to allow users to maximise the use out of their Ultra-Long DNA Sequencing Kit V14.
The EEB Expansion (EXP-EEB001) contains enough reagents for at least 6 standard extraction elution steps.
The Ultra-Long Auxiliary Vials (EXP-ULA001) provides enough reagents to carry out twelve additional flow cell loads on MinION or PromethION flow cells.
EEB Expansion (EXP-EEB001) contents:
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Extraction EB EEB White 1 6,000 Ultra-Long Auxiliary Vials (EXP-ULA001) contents:
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Elution Buffer EB Black 1 1,500 Sequencing Buffer UL SBU Red 2 1,000 Loading Solution UL LSU White cap, pink label 1 200 Flush Tether UL FTU Purple 1 600 Flow Cell Flush FCF Clear cap, light blue label 1 15,500