- Materials
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- 1200 ng gDNA per sample
- Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL)
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
- Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
- Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
- Hamilton PCR ComfortLid (Cat# 814300)
- Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)
- Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
- Equipment
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- P100 pipette and tips
- Qubit fluorometer (or equivalent)
- Ice bucket with ice
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Users have the option to run select steps in the protocol. We recommend quantification after End Repair for barcode balancing.
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Consumables and equipment quantities:
Consumables No. of consumables for all conditions Hamilton 50 µl CO-RE tips with filter 960 Hamilton 300 µl CO-RE tips with filter 960 Hamilton 1000 µ CO-RE tips with filter 96 Bio-Rad Hard-Shell® 96-well PCR Plate 8 Note: We recommend using Hamilton tips for efficient liquid handling.
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Reagent quantities:
Note: Volumes for x48 samples will be available soon.
Multiple steps:
End Repair and Adenylation step to Native Barcode Ligation step
Reagents x96 samples 80% ethanol 80 ml AMPure XP Beads 15 ml Nuclease-free water 10 ml NEBNext FFRE DNA Repair Buffer 130 µl NEBNext FFPE DNA Repair Mix 90 µl Ultra II End Prep Reaction Buffer 130 µl Ultra II End Prep Enzyme Mix 120 µl Blunt/TA Ligase Master Mix 1210 µl EDTA 1 vial Native Barcode plate 1 plate Native Barcode Ligation step to Adapter Ligation step
Reagents x96 samples 80% ethanol 40 ml AMPure XP Beads 15 ml Nuclease-free water 10 ml Long Fragment Buffer (LFB) 2 bottles Elution Buffer (EB) 1 bottle Blunt/TA Ligase Master Mix 1210 µl EDTA 1 vial Native Barcode plate 1 plate Adapter Mix F (AMII F) 1 vial NEBNext Quick Ligation Reaction Buffer (5x) 590 µl Quick T4 DNA Ligase 310 µl
Individual steps:End Repair step
Reagents x96 samples 80% ethanol 40 ml AMPure XP Beads 15 ml Nuclease-free water 10 ml NEBNext FFRE DNA Repair Buffer 130 µl NEBNext FFPE DNA Repair Mix 90 µl Ultra II End Prep Reaction Buffer 130 µl Ultra II End Prep Enzyme Mix 120 µl Native Barcode Ligation step
Reagents x96 samples 80% ethanol 40 ml AMPure XP Beads 15 ml Nuclease-free water 10 ml Blunt/TA Ligase Master Mix 1210 µl EDTA 1 vial Native Barcode plate 1 plate Adapter Ligation step
Reagents x96 samples AMPure XP Beads 15 ml Long Fragment Buffer (LFB) 2 bottles Elution Buffer (EB) 1 bottle NEBNext Quick Ligation Reaction Buffer (5x) 590 µl Quick T4 DNA Ligase 310 µl -
Prepare the reagents as follows and store on ice.
Reagent 1. Thaw at room temperature 2. Briefly spin down NEBNext FFPE DNA Repair Buffer ✓ - NEBNext FFPE DNA Repair Mix ✓ Note: Do no vortex NEBNext Ultra II End-prep repair buffer ✓ - NEBNext Ultra II End-prep enzyme mix ✓ Note: Do not vortex NEBNext Quick Ligation Reaction Buffer (5x) ✓ - NEBNext Quick T4 DNA Ligase ✓ Note: Do not vortex Native barcode plate ✓ ✓ Elution Buffer (EB) ✓ - Adapter Mix II F (AMII-F) ✓ ✓ Long Fragment Buffer (LFB) ✓ - -
In a clean hard shell PCR plate, prepare the sample input plate as follows:
- Dispense 1200 ng DNA into each sample well. Note: We suggest aliquoting your DNA at 80 ng/µl per sample.
- Make up the volume of each well containing DNA samples to at least 15 µl.
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Switch on the Hamilton NGS STAR 96 robot and open the method from the desktop shortcut.
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When the method is loaded, click 'Start'.
To find further information, click 'About MLK111.96-XL' to view the automation section of the Community in the default web browser.
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Click 'MLK111.96-XL' to proceed to the method parameter selection.
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Optional actionBefore starting, a user ID can be entered for traceability purposes.
Note: Any format of user ID can be used.
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Enter the barcode of the input plate containing the samples and the output plate which will contain the prepared DNA libraries.
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Optional actionSelect where to start on a previously used native barcode plate when using 48 or fewer samples.
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Click "Select steps" and click "Ok".
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To perform a singular step, check "Perform only selected step" and click "Ok".
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To perform multiple steps, leave the check box unselected and click "Ok".
Users will only be able to select a step that canonically comes after the first step selected.
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Once settings and the steps for the run have been selected, there will be a series of dialogues illustrating how to load the deck depending on the steps selected.
For an example of the dialogues illustrating how to load the deck, please see the "Complete automated library preparation" step.
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Optional actionIf quantities allow, the libraries may be diluted in Elution Buffer (EB) for splittling across multiple flow cells.