为获得最优表现，NEB建议如下：

1. 于冰上解冻所有试剂。

2. 轻弹并/或翻转各管，确保各试剂充分混匀。
   注意： 请切勿涡旋振荡 FFPE DNA修复混合液或 Ultra II末端修复酶混合物。

3. 同一日内首次打开一管试剂前，请务必先将该管试剂瞬时离心。

4. Ultra II 末端修复缓冲液和 FFPE DNA 修复缓冲液内可能出现少量沉淀。待此两管液体回复至室温后，使用移液枪上下吹打数次，打散沉淀；然后涡旋振荡30秒，以确保沉淀充分溶解。
   注意： 请务必涡旋振荡混匀缓冲液。

5. FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。

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'.

文库片段长度	测序芯片上样量
非常短 (<1 kb)	100 fmol
短 (1-10 kb)	35–50 fmol
长 (>10 kb)	300 ng

请注意： 如您的文库产量低于我们的推荐值，请使用全部文库上样。

您可按需使用质量与摩尔数转换计算器，如 NEB 计算器。

请注意： 我们正在将部分试剂的包装形式由单次管装改为瓶装。请按照与您所用试剂盒包装相对应的说明操作。

单次使用管装： 向一整管测序芯片冲洗液（FCF）中加入30µl 测序芯片系绳（FCT）。

瓶装： 请另拿一支适当体积的离心管制备测序芯片预处理液：

试剂	体积（每张芯片）
测序芯片冲洗液 (FCF)	1,170 µl
测序芯片系绳 (FCT)	30 µl
总体积	1,200 µl

1. 将测序芯片平放在金属板上。

2. 将测序芯片推入对接端口，直至金色引脚或绿色电路板不可见。

1. 将测序芯片与连接器横竖对齐，以便顺利卡入。

2. 用力下压芯片至卡槽，并确认卡夹位置归位。

1. 将P1000移液枪转至200µl刻度。
2. 将枪头垂直插入加液孔中。
3. 反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

1. 将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
2. 用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

您可在纳米孔社区获取 测序芯片清洗试剂盒实验指南。

您可在 此处找到回收测序芯片的说明。

请注意： 在将测序芯片寄回之前，请使用去离子水对每张芯片进行冲洗。