Hieff™ LongSeq Amplicon End Repair and Ligation Module is a library preparation kit specifically developed for the third-generation Nanopore sequencing platform. When used in combination with 13304ES, it enables library construction for PCR products of various lengths. This kit is designed for amplicon library preparation. It features a streamlined workflow where the product undergoes end repair and A-tailing without the need for intermediate purification, allowing for direct barcode ligation. This makes the experimental procedure more convenient. Compatible with input amounts ranging from 50 ng to 1.5 µg. Library preparation time is approximately 2 hours. Stringent quality control for batch-to-batch performance and stability. Specifications Product Name Cat.NO. Specification Hieff™ LongSeq Amplicon End Repair and Ligation ModuleAmplicon Library Preparation Module 13306ES08 8 T 13306ES24 24 T 13306ES96 96 T Components Components No. Name 13306ES08 13306ES24 13306ES96 8 T 24 T 96 T 13306-A Endprep Buffer 56 μL 168 μL 672 μL 13306-B Endprep Enzyme 24 μL 72 μL 288 μL 13306-C Rapid Ligase Master Mix 280 μL 840 μL 4×840 μL Instructions 1. Materials to be Prepared by User 1) DNA Quantification: 1× dsDNA HS Assay Kit (Cat# 12642ES); Qubit Assay Tubes (Cat# 83509ES); or equivalent products from other vendors. 2) DNA Quality Control: TapeStation DNA ScreenTape or other equivalent instruments. 3) Barcode Adapter: Hieff™ Native Barcode Kit for ONT, Set 1 (Cat# 13317) Hieff™ Native Barcode Kit for ONT, Set 2 (Cat# 13318) Hieff™ Native Barcode Kit for ONT, Set 3 (Cat# 13319) Hieff™ Native Barcode Kit for ONT, Set 4 (Cat# 13320) Native Barcoding Expansion 1-12 (Nanopore #EXP-NBD104) Native Barcoding Expansion 13-24 (Nanopore #EXP-NBD114) Native Barcoding Expansion 1-96 (Nanopore #EXP-NBD196) 4) Motor Protein Adapter: Adapter Mix II Expansion (Nanopore #EXP-AMII001) Native Adapter (Nanopore #EXP-NBA114) 5) Motor Protein Ligation Module and Purification Beads: Hieff™ Adapter Ligation Module for ONT (Cat# 13304), Hieff NGS™ DNA Selection Beads V2 (Cat# 12418), AMPure XP Beads (Beckman), or equivalent products from other vendors. 6) Nucleic Acid Purification Buffers: Additional Long Fragment Buffer (LFB) (Nanopore #EXP-LFB001) Additional Short Fragment Buffer (SFB) (Nanopore #EXP-SFB001) 7) Other Materials: Freshly prepared 80% ethanol, Nuclease-free ddH₂O, 0.5 M EDTA, TE Buffer (10 mM Tris-HCl, pH 8.0~8.5 + 1 mM EDTA), low-binding EP tubes, PCR tubes, wide-bore pipette tips, magnetic rack, thermal cycler, and vortex mixer, etc. 2. Workflow Figure 1. Library Preparation Workflow for PCR Products 3. Protocol 3.1 End Repair / dA-Tailing This step is used for DNA end blunting, 5' phosphorylation, and 3' dA-tailing. 1) Thaw Endprep Buffer and Endprep Enzyme on ice. Mix by inversion, briefly centrifuge, and keep on ice. Prepare the reaction mixture in a PCR tube according to the volumes specified in Table 1. Table 1. Reaction System for Enzymatic Digestion / End Repair / dA-Tailing Name Volume (μL) Input DNA x Endprep Buffer 7 Endprep Enzyme 3 ddH₂O Up to 60 2) Mix gently by pipetting (do not vortex) and briefly centrifuge to collect the reaction mixture at the bottom of the tube. 3) Place the PCR tube in a thermal cycler and run the program set in Table 2: Table 2. PCR Program for End Repair / dA-Tailing Temperature Time Heat Lid 105℃ On 30℃ 10 min 72℃ 10 min 4℃ Hold 3.2 Native Barcode Ligation This step is used to ligate Native Barcodes to both ends of the product from Step 3.1. 1) Thaw Native Barcode and Rapid Ligase Master Mix on ice. Mix by flicking, briefly centrifuge, and keep on ice. Prepare the reaction mixture in a PCR tube according to the volumes specified in Table 3. Table 3. Reaction System for Native Barcode Ligation Name Volume (μL) Product from Step 3.1 60 Native Barcode* 2.5 Rapid Ligase Master Mix 35 ddH₂O Up to 100 [Note]: We recommend using the Yesea Associated products (Cat# 13317~13320) or ONT designated products (Nanopore #EXP-NBD104/EXP-NBD114/EXP-NBD196) for Native Barcode. 2) Mix gently by pipetting (do not vortex) and briefly centrifuge to collect the reaction mixture at the bottom of the tube. 3) Place the PCR tube in a thermal cycler and run the program set in Table 4: Table 4. PCR Program for Native Barcode Ligation Temperature Time Heat Lid 105℃ Off 20℃ 20 min 4℃ Hold 3.3 Termination of Native Barcode Ligation (Optional) This step uses EDTA to terminate the ligation reaction from Step 3.2. Termination is required before pooling samples; failure to terminate before pooling may result in cross-ligation between different groups of Barcodes. Termination is optional for single-tube purification. 1) Preparation: Prepare 0.5 M EDTA in advance. 2) Add 4 μL of 0.5 M EDTA to the Native Barcode ligation product, mix thoroughly, and proceed to the purification step. 3.4 Purification of Native Barcode Ligation Product This step purifies the product from Step 3.2 or 3.3 using Hieff NGS™ DNA Selection Beads V2 (Cat# 12418). 1) Preparation: Remove the Hieff NGS™ DNA Selection Beads and allow them to equilibrate at room temperature for 30 minutes. Prepare 80% ethanol. 2) Vortex or invert the magnetic beads thoroughly to ensure homogeneity before use. 3) For libraries >1 Kb, add 40 μL of Hieff NGS™ DNA Selection Beads V2 (0.4×, Beads:DNA = 0.4:1) to the product from Step 3.2 or 3.3. Mix by pipetting 10 times and incubate at room temperature for 10 minutes. For libraries 1 Kb, add 40 μL of Hieff NGS™ DNA Selection Beads V2 (0.4×, Beads:DNA = 0.4:1) to the product from Step 3.5. Mix by pipetting 10 times and incubate at room temperature for 10 minutes. For libraries
