MR300D Standardized NGS DNA Fragmentation Workflow

MR300D Standardized NGS DNA Fragmentation Workflow

1. Purpose and Scope

DNA fragmentation is a key upstream step in NGS library preparation. This workflow uses the MR300D ultrasonic DNA shearing system to establish repeatable non-contact fragmentation conditions for genomic DNA, cfDNA, plasmid DNA, and microbial DNA. It supports common library targets including 170 bp, 350 bp, 500 bp, and 800 bp while keeping sample handling, temperature control, QC, and troubleshooting traceable.

2. Equipment and Sample Requirements

• Equipment: MR300D non-contact ultrasonic DNA shearing system with low-temperature water circulation.
• Supporting instruments: Qubit or Nanodrop, agarose gel electrophoresis, fragment analyzer, refrigerated centrifuge, and clean bench.
• Consumables: nuclease-free 0.5 ml or PCR thin-wall tubes, nuclease-free water, NGS elution buffer, ice box, and tube rack.
• Sample entry criteria: Qubit concentration above 20 ng/ul, OD260/280 around 1.8-2.0, OD260/230 around 2.0-2.2, no visible degradation, no repeated freeze-thaw cycles, and no turbidity or precipitation.

3. Standard Workflow

1. UV-sterilize the work area and pre-cool the MR300D and circulation system to 4 C.
2. Mix DNA gently, briefly centrifuge, and prepare a 100 ul shearing system according to sample concentration.
3. Remove bubbles and place tubes symmetrically in the rack. Empty rack positions should be balanced when needed.
4. Select the target fragment condition and start non-contact ultrasonic processing.
5. Briefly centrifuge after processing, keep samples on ice, and proceed to library construction or QC immediately.

4. Reference Fragmentation Conditions

Target fragment	Amplitude	Total time	Pulse mode	Use case
170 bp	50%	900 s	15 s on / 15 s off	Targeted sequencing and ctDNA
350 bp	30%	600 s	15 s on / 15 s off	Standard WGS and WES libraries
500 bp	30%	450 s	15 s on / 15 s off	Low-depth WGS
800 bp	30%	300 s	15 s on / 15 s off	Long-fragment enrichment and microbial genomes

5. QC and Troubleshooting

Run agarose gel screening and, when needed, fragment analyzer QC. Qualified samples should show concentrated bands, no high-molecular-weight residue, no severe smear, and a main peak close to the target range. If fragments remain too large, extend processing time by 50-100 s, increase amplitude cautiously, remove bubbles, and verify temperature control. If fragments are too small, reduce amplitude or total time and check whether the sample experienced freeze-thaw damage.

6. Summary

MR300D standardizes NGS DNA fragmentation by combining non-contact ultrasonic processing, 4 C temperature control, traceable parameters, and post-shearing QC. The workflow is suitable for method setup, batch processing, and routine library preparation support.