For optimal performance, NEB recommend the following:

1. Thaw all reagents on ice.

2. Flick and/or invert the reagent tubes to ensure they are well mixed.
   Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.

3. Always spin down tubes before opening for the first time each day.

4. The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
   Note: It is important the buffers are mixed well by vortexing.

5. The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

Note: Any number of samples between 8-96 can be processed using this protocol.

• Please note that if removing the sample plate for off-deck storage, user interaction is required approximately 10 minutes after starting the run.
• We recommend room temperature for the majority of users. However, 37°C can be beneficial for recovery of longer DNA strands.
• The MinION option is valid for both the MinION and GridION device.

Note: Please note only the ONT recommended settings have been verified.

• The required loading position for each box will flash to indicate where to place the labware.
• Please load full tip boxes only, partially full boxes of filtered tips for the Multichannel Arm 384/96 are currently not supported.
• Click 'Approve' after each addition of labware to proceed to the next box.
• After loading all of the required labware, close the front safety shield and click 'Next Page' to proceed.

• The required loading position for each box will flash to indicate where to place the labware.
• Please load full tip boxes only, partially full boxes of filtered tips for the Multichannel Arm 384/96 are currently not supported.
• Click 'Approve' after each addition of labware to proceed to the next box.
• After loading all of the required labware, close the front safety shield and click 'Next Page' to proceed.

• The required loading position for each box will flash to indicate where to place the labware.
• Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
• Click 'Approve' after each addition of labware to proceed to the next box.

• The required loading position for each box will flash to indicate where to place the labware.
• Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
• Click 'Approve' after each addition of labware to proceed to the next box.

• The required loading position for each box will flash to indicate where to place the labware.
• Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
• Click 'Approve' after each addition of labware to proceed to the next box.
• After loading all of the required labware, click 'Next Page' to proceed.

• The required loading position will flash to indicate where to place the labware.
• Click 'Approve' after the addition of the metal lid for ODTC to proceed.

Loading the metal lid for ODTC:

Note: Take care to position the metal lid centrally in the recess. Incorrect positioning of the metal lid can lead to run error.

• The required loading position will flash to indicate where to place the labware.
• After loading the Waste plate, click 'Next Page'.

Loading the Waste Plate:

Incorrect positioning of the reaction plates will result in incorrect sample tracking throughout the run. Please ensure the reaction plates are placed on the worktable in the correct orientation:

• For the reaction plate(s) loaded in the 'Hotel': The lettered well markers (A-H) should be positioned towards the back of the worktable and the numbered well markers (1-12) should be positioned facing the right.

• For the reaction plate(s) loaded onto the worktable: Follow standard plate orientation, with the lettered well markers (A-H) positioned to the left and the numbered well markers (1-12) positioned to towards the back of the worktable.

  
  __Note:__ We recommend all plates are labelled before placing on the worktable to ensure correct plate tracking.
  

• The required loading position for each plate will flash on the on-screen display to indicate where to place the labware.

• Click 'Approve' after each addition of labware to proceed.

• After loading all of the required labware, click 'Next Page'.

• The required loading position will flash to indicate where to place the labware.
• Click 'Approve' after each addition of labware to proceed.
• After loading all of the required labware, click 'Next Page'.

Note: We recommend all plates are labelled before placing on the worktable to ensure correct plate tracking.

Loading the 'Bead plate':

Loading the 'Elution buffer plate':

Loading the 'Ethanol plate':

We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.

• Ensure the master mix is homogenous by pipette mixing.
• Avoid the introduction of air bubbles while preparing the master mix.
• Avoid leaving droplets on the tube wall.
• Briefly spin down the End-prep (EP) master mix to ensure all the liquid is at the bottom of the Sarstedt tube.

Reagent volumes for all sample numbers:

Reagent	Volume per sample	Volume X8 samples	Volume X24 samples	Volume X48 samples	Volume X96 samples
NEBNext FFPE DNA Repair Buffer	3.5 µl	36.4 µl	109.2 µl	201.6 µl	369.6 µl
NEBNext FFPE DNA Repair Mix	2 µl	20.8 µl	62.4 µl	115.2 µl	211.2 µl
Ultra II End-prep reaction buffer	3.5 µl	36.4 µl	109.2 µl	201.6 µl	369.6 µl
Ultra II End-prep enzyme mix	3 µl	31.2 µl	93.6 µl	172.8 µl	316.8 µl
Total	12 µl	124.8 µl	374.4 µl	691.2 µl	1267.2 µl

Note: Reagent volumes will vary in accordance with the number of samples selected for processing (8-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes. Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.

We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.

• Ensure the master mix is homogenous by pipette mixing.
• Avoid the introduction of air bubbles while preparing the Adapter-ligation (AL) master mix.
• Avoid leaving droplets on the tube wall.
• Briefly spin down the Adapter-ligation (AL) master mix to ensure all the liquid is at the bottom of the Sarstedt tube(s).

Reagent volumes for preparation in each tube for all sample numbers:

Reagent	Volume per sample	Volume X8 samples	Volume X24 samples	Volume X48 samples	Volume X96 samples
Number of tubes to prepare	-	1	1	2	4
Ligation Buffer (LNB)	25 µl	260 µl	780 µl	660 µl	660 µl
NEBNext Quick T4 DNA Ligase	10 µl	104 µl	312 µl	264 µl	264 µl
Ligation Adapter (LA)	5 µl	52 µl	156 µl	132 µl	132 µl
Total volume in each tube	-	416 µl	1,248 µl	1,056 µl	1,056 µl
Total volume prepared	-	416 µl	1,248 µl	2,112 µl	4,224 µl
Note: Reagent volumes will vary in accordance with the number of samples selected for processing (8-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes and split accordingly across the Sarstedt tube(s).
Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.

• Ensure the master mixes are thoroughly mixed before loading.
• You will need to select each loading position using the Tecan's TouchTools touchscreen display.
• Follow the instructions on the display for each reagent, ensuring the fill volume for each tube is correct.
• Ensure the reagents have been added to all of the required positions before proceeding.
• Click 'Confirm' to proceed.

On-screen abbreviation glossary:

• End-prep master mix – EP
• Adapter ligation master mix – AL

• Ensure all the reagents have been thoroughly mixed by vortexing before dispensing into the troughs.
• The required loading position will flash to indicate where to insert the trough.
• Click 'Approve' after each addition to proceed.
• After loading all of the troughs, click 'Next Page'.

__Note:__ Follow the on-screen instructions for the reagent fill volume and the required trough to use.

• Site 2: Trough with fresh 80% ethanol.
• Site 3: Trough with AMPure XP Beads.
• Site 5: Trough with Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB), depending on use.
• Site 6: Trough with Elution Buffer (EB).
• Site 7: Trough with nuclease-free water.

• Quantify your sample input using a Qubit fluorometer (or equivalent).
• Per sample, transfer 1 μg (or 100-200 fmol) of input DNA into a well of the input plate.
• Adjust the volume to 60 μl with nuclease-free water.
• Mix thoroughly by pipetting.
• Spin down briefly in a microfuge.
• The required loading position will flash to indicate where to place the labware.
• Click 'Approve' after the addition of labware to proceed.
• After loading all of the required labware, click 'Next Page'.

Fragment library length	Flow cell loading amount
Very short (<1 kb)	100 fmol
Short (1-10 kb)	35–50 fmol
Long (>10 kb)	300 ng

Note: If the library yields are below the input recommendations, load the entire library.

If required, we recommend using a mass to mol calculator such as the NEB calculator.

Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.

Single-use tubes format: Add 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).

Bottle format: In a clean suitable tube for the number of flow cells, combine the following reagents:

Reagent	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

1. Place the flow cell flat on the metal plate.

2. Slide the flow cell into the docking port until the gold pins or green board cannot be seen.

1. Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
2. Press down firmly onto the flow cell and ensure the latch engages and clicks into place.

1. Set a P1000 pipette tip to 200 µl.
2. Insert the tip into the inlet port.
3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

1. Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
2. Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Dispose of the used tips in an appropriate container.

Note: Take care not to spill any residual liquid waste when removing from the worktable.

• Wipe the lid with 10% bleach
• Thoroughly rinse the bleach off the lid using ≥80% ethanol or double distilled water (ddH2O) and lint-free wipes
• Allow the lid to air dry

• Remove all Flexible Channel Arm (FCA) hanging tip trays from the FCA standard tip carriers, and discard accordingly.
• Remove all empty MultiChannel Arm (MCA) tip boxes from the worktable and discard accordingly.
• For partially used tip boxes, consider restacking the box with the appropriate tips for the next run.

Note: The MultiChannel Arm (MCA) tip boxes must be fully stacked. Failure to fully stack the MCA tip boxes can result in run error.

The Flow Cell Wash Kit protocol is available on the Nanopore Community.

Instructions for returning flow cells can be found here.

Note: All flow cells must be flushed with deionised water before returning the product.