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'.

フラグメントライブラリーの長さ	フローセルへのロード量
非常に短い(<1 kb)	100 fmol
短い (1-10 kb)	35–50 fmol
長い (>10 kb)	300 ng

注）: ライブラリーの収量が推奨入力値以下の場合は、ライブラリー全体をロードしてください。

必要であれば、mass-to-mol計算機 NEB calculatorの使用をお勧めします。

注） 現在、使い捨てチューブを使用したキットをボトル型にフォーマット変更中です。ご使用のキットのフォーマットに従ってください。

シングルユースチューブの場合: 30μlのFlow Cell Tether（FCT）をFlow Cell Flush（FCF）チューブに直接加えてください。

ボトルフォーマットの場合: フローセルの数に適したチューブに、以下の試薬を入れてくださいs:

試薬	フローセルあたりの容量
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
合計	1,200 µl

1. フローセルを金属プレートの上に平らに置きます。

2. フローセルを、金色のピンまたは緑色の基板が見えなくなるまでドッキングポートにスライドさせます。

1. フローセルとコネクターを水平および垂直に並べてから、所定の位置にスムーズに挿入してください。
2. フローセルをしっかりと押し下げ、ラッチがかみ合い、カチッと音がして所定の位置に収まることを確認します。

1. P1000ピペットチップを200µlにセットします。
2. チップをインレットポートに挿入します。
3. ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファ ーが入るのが確認できるまで、ホイールを回します。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注） アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

1. ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
2. ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

Flow Cell Wash Kit protocolは、Nanoporeコミュニティーで入手できます。

フローセルの返却方法は hereをご覧ください。

(注： 製品を返却する前に、すべてのフローセルを脱イオン水で洗浄する必要があります。