Hieff™ Adapter Ligation Module is a Motor Protein Ligation module specifically developed for the third-generation sequencing platform. This kit features engineered and optimized enzymes and buffers, enabling the efficient ligation of sequencing adapters containing Motor Protein to both ends of the target DNA fragments. It can be used in conjunction with the Hieff™ DNA Endprep & dA-Tail Module (Cat#13302) and the Hieff NGS™ Barcode Ligation Module(Cat#13303) for library preparation on the Nanopore platform. All reagents have undergone rigorous quality control and functional validation to ensure the stability and reproducibility of library construction. Specifications Cat.No. 13304ES08 / 13304ES24 / 13304ES96 Size 8 T / 24 T / 96 T Components Components No. Name 13304ES08 13304ES24 13304ES96 8 T 24 T 96 T 13304-A Rapid Ligation Reaction Buffer（5×） 160 μL 480 μL 2×960 μL 13304-B Rapid T4 DNA Ligase 40 μL 120 μL 480 μL Instructions 1. Materials to be Prepared by User 1) Sample Fragmentation: g-Tube tube fragmentation, Megaruptor 3 System (Diagenode), or other equivalent fragmentation methods; 2) DNA Quantification: 1× dsDNA HS Assay Kit (Cat# 12642ES); Qubit dedicated assay tubes (Cat# 83509ES); or equivalent products from other manufacturers; 3) DNA Quality Control: TapeStation DNA ScreenTape or other equivalent instruments, etc.; 4) Barcode Adapter: Native Barcoding Expansion 1-12 (Nanopore #EXP-NBD104); Native Barcoding Expansion 13-24 (Nanopore #EXP-NBD114); Native Barcoding Expansion 1-96 (Nanopore #EXP-NBD196); 5) Motor Protein Adapter: Adapter Mix II Expansion (Nanopore #EXP-AMII001) Native Adapter (Nanopore #EXP-NBA114); 6) Purification Magnetic Beads: Hieff NGS™ DNA Selection Beads (Cat# 12601), AMPure XP Beads (Beckman) or equivalent products from other manufacturers; 7) Nucleic Acid Purification Buffers: Additional Long Fragment Buffer (LFB) (Nanopore #EXP-LFB001); Additional Short Fragment Buffer (SFB) (Nanopore #EXP-SFB001); 8) Other Materials: Freshly prepared 80% ethanol, Nuclease-free ddH2O, TE Buffer (10 mM Tris-HCl, pH 8.0~8.5 + 1 mM EDTA); low-adsorption EP tubes, PCR tubes; wide-bore pipette tips, magnetic rack, thermal cycler, and vortex mixer, etc. 2. Procedure This step is used for Native Adapter Ligation of the Barcode Adapter product. 1) Take Rapid Ligation Reaction Buffer (5×), Rapid T4 DNA Ligase, and Native Adapter thawed on ice, mix by inversion, briefly centrifuge, and place on ice. Prepare the reaction system according to the required volumes of each component in Table 1: Table 1. PCR Reaction System for Native Adapter Ligation Name Volume（μL） Purified Product of Native Barcode Ligation 30 Rapid Ligation Reaction Buffer (5×) 20 Native Adapter (NA)* 5 Rapid T4 DNA Ligase 5 ddH2O Up to 100 [Note]: We recommend using the ONT designated product for Native Adapter (NA) (Nanopore #EXP-NBA114). 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 tubes into a thermal cycler, set the program according to Table 2, and perform the Native Adapter ligation reaction. Table 2. PCR Program for Native Adapter Ligation Reaction Temperature Time Heat Lid Off 20℃ 20 min 4℃ Hold Purification of Native Adapter Ligation Product This step purifies the reaction product using Hieff NGS™ DNA Selection Beads (Cat# 12601). 1) Preparation: Remove the Hieff NGS™ DNA Selection Beads and allow them to equilibrate at room temperature for 30 minutes. Remove the Nanopore-compatible nucleic acid wash buffer. 2) Vortex or invert the magnetic beads thoroughly to ensure homogeneity. 3) Add 40 μL of Hieff NGS™ DNA Selection Beads (0.4×, Beads : DNA = 0.4 : 1) to the reaction product from Step 3.4. Mix gently by pipetting 10 times and incubate at room temperature for 10 minutes. 4) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads from the liquid. After the solution clears (approx. 5 min), carefully remove the supernatant. 5) Add 200 μL of Long Fragment Buffer (LFB)* or Short Fragment Buffer (SFB)** to resuspend the beads (Do not use 80% ethanol!!!). Mix thoroughly by pipetting 10 times. Return the PCR tube to the magnetic rack. After the solution clears, remove the supernatant. (The volume of LFB or SFB can be adjusted based on your previous experimental conditions!) *If enriching for DNA fragments of 3 kb or longer, we recommend using LFB (Nanopore #EXP-NBA114); **If aiming to retain a broader range of DNA fragment lengths, we recommend using SFB (Nanopore #EXP-NBA114). 6) Repeat Step 5 for a total of two washes. 7) Place the PCR tube on the magnetic rack and remove the residual supernatant. 8) Remove the PCR tube from the magnetic rack. Add 18 μL of EB buffer or elution buffer (10 mM Tris-HCl, pH 8.0-8.5) to cover the beads. Pipette to mix. Incubate at 37°C for 10 minutes. If the beads are dry or cracked, shorten the incubation time appropriately. 9) Place the PCR tube on the magnetic rack to separate the beads from the liquid until the solution clears (approx. 5 min). 10) Transfer 16 μL of the supernatant to a new PCR tube. Take 1 μL for Qubit quantification. Store the library at 4°C. Notes 1. For your safety and health, please wear a lab coat and disposable gloves when handling this product. 2. Thaw all kit components on ice prior to use. Invert the tubes several times to mix thoroughly, briefly centrifuge, and then place on ice until ready for use. 3. We recommend mixing reaction solutions by pipetting. Avoid vigorous vortexing, as it may reduce library yield. 4. To prevent cross-contamination, we recommend using filter tips and changing pipette tips when handling different samples. 5. We recommend performing all reactions in a thermal cycler with a heated lid. Please preheat the instrument to the target reaction temperature beforehand. 6. Do not mix reagents from different lot numbers. 7. For research use only!
