Nanodigmbio-NadPrep Hybrid Capture Reagents
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DNA Libraries (for Illumina®) User Manual V2.5

Please refer to the latest version from Nanodigmbio (http://www.njnad.com/download-protocols/). This instruction is intended for use with the NAD Panels and Blockers on Illumina® platforms. For research use only. Not for use in diagnostic procedures. Nanodigmbio (Nanjing) Biotechnology Co., Ltd. reserves all rights. Revision History Version Data Released Revise description of storage temperatures for kit and components. Updated kit contents of NadPrep® Hybrid Capture Reagents. Updated“NadPrep”as the official trademark. Updated recommended products and company information. Updated description...

NadPrep NanoBlockers (for Illumina®) NadPrep Hybrid Capture Reagents Library Input Step 1: Perform Hybridization Reaction Step 4: Purify and Quantify Library Plate Protocol Step 1: Perform Hybridization Reaction Step 4: Purify and Quantify Library Nanodigmbio (Nanjing) Biotechnology Co., Ltd.

NAD Panels within 5'-biotinylated probes are predesigned and optimized for targeted capture applications in next generation sequencing. Panels can be upgraded by either expanding with spike-in probes or combining with other panels. If you plan to use NAD Panels as spike-in panels, contact us (support@njnad.com) for professional and specific recommendations. NadPrep NanoBlockers (for Illumina®) are universal blockers for Illumina® platforms. The NadPrep NanoBlockers act by reducing non-specific binding of adapter sequences, which improves on-target rate and enrichment efficiency. NadPrep...

Library Input Consideration Library Preparation This protocol was verified with libraries prepared by NadPrep DNA Universal Library Preparation Kit (for Illumina®) and NadPrep cfDNA Library Preparation Kit (for Illumina®). Please adjust the length of fragmented DNA to match sequencing mode. Library Input For genome DNA, 500 ng of each library for hybrid capture is recommended. In most cases, multiplexing up to 12 samples (6 µg of total DNA) has limited impact on capture performance. A vacuum concentrator is recommended for concentrating DNA. Although the beads-based concentration method...

Item Description Digital electrophoresis Agilent 2100 Electrophoresis Bioanalyzer® system (Cat # G2939AA) Agilent 2200 Electrophoresis Bioanalyzer® system (Cat # G2965AA) Bioptic Qsep100 capillary gel electrophoresis system or equivalent Pipettor General laboratory supplier Thermal cycler General laboratory supplier Benchtop centrifuge General laboratory supplier Microcentrifuge General laboratory supplier Vortex mixer General laboratory supplier Magnet stand Tube protocol: Thermo Fisher DynaMag™ -96 Side Magnet (Cat # 12331D) Thermo Fisher DynaMag™ -PCR Magnet (Cat # 492025) or equivalent...

NadPrep NanoBlockers (for Illumina®) NadPrep NanoBlockers (for Illumina®), 16 rxn 1006101 -25 ~-15°C NadPrep NanoBlockers (for Illumina®), 96 rxn 1006102 -25 ~-15°C Absolute ethanol General laboratory supplier, analytical grade or equivalent (10 mM Tris, 0.1 mM EDTA) Purification reagents Beckman-Coulter Agencourt® AMPure® XP - PCR Purification beads (Cat # A63880) NadPrep SP Beads or equivalent Kapa Biosystems HiFi HotStart ReadyMix (Cat # KK2601) NadPrep 2X HiFi PCR Master Mix NadPrep Amplification Primer Mix (for Illumina®) (Cat # 1004101/1004102) or equivalent PCR Plate Sealing Film...

Perform Hybridization Concentrate libraries Perform hybridization reaction Perform Wash Prepare buffers Wash streptavidin beads Perform washes Perform bead capture Purify and Quantify Library * Perform during hybridization reaction. Safe stopping point Nanodigmbio (Nanjing) Biotechnology Co., Ltd.

This protocol is validated by the Exome Plus Panel v2.0 (from Nanodigmbio). 1. Thaw Hyb #1 and Hyb #2 of NadPrep Hybrid Capture Reagents to room temperature. o Note: Inspect the tube of Hyb #1 for possible crystallization of salts. If crystals exist, heat the tube up to 65°C with shaking occasionally until the solution is completely solubilized. The heating process may take minutes to hours. 2. When using a vacuum concentrator for DNA concentration, please preheat the instrument and adjust the temperature to 60°C. o Note: Considering no GC bias, we highly recommend using vacuum system for...

7. Thaw Exome Plus Panel v2.0 on ice. 8. Prepare the Hybridization Master Mix in the tube from step 6 as follows: Exome Plus Panel v2.0 4 |iL 9. Pipet the mixture for 15-20 times. Briefly centrifuge and incubate the tube at 25°C for 5-10 min. 10. Vortex and centrifuge to collect the contents. Transfer 17 |iL of the solution to a new low-bind 0.2 mL PCR tube. 11. Incubate the tube in the thermal cycler programmed as follows: o *4 hr incubation has no compromise on the performance of Exome Plus Panel v2.0 (>1 Mb region). For GC-rich regions or small panels (<1 Mb region), longer incubation...

1. Thaw all wash buffers of NadPrep Hybrid Capture Reagents to room temperature, mix thoroughly and centrifuge to collect the contents. © Note: If Wash Buffer 1 does not thaw sufficiently, heat the bottle at 65°C in a water bath/heating block until the solution is clear. 2. Equilibrate the streptavidin beads to room temperature for a minimum of 30 min prior to use. 3. Aliquot the following Wash buffers into low-bind tubes. BeadWashBuffer 320 |iL Keep at room temperature. Wash Buffer 1 280 |iL Aliquot 110 |iL of the Buffer into a separate tube and heat to 65°C. The remaining solution should...

Perform Capture Reaction 1. After the hybridization reaction (4‒16 hr), initiate the Wash program immediately. 2. Transfer the resuspended preheated streptavidin beads to the 0.2 mL tube containing the library. Briefly vortex until the beads are fully resuspended. Note: Operate quickly by using barrier pipette tips. 3. Place the sample tube in the thermal cycler and incubate at 65℃ for 45 min. Briefly vortex the tube out of the thermal cycler to resuspend the beads for every 10-12 min. Heated Washes Note: Operate quickly and be careful to minimize bubble formation during pipetting. 1. After...