ONT Midnight RT PCR Expansion kit, 576/kit

The Oxford Nanopore Technology (ONT) Midnight RT PCR Expansion Kit is a highly efficient solution for whole-genome sequencing of SARS-CoV-2. This kit, designed for use with ONT’s Rapid Barcoding Kits (SQK-RBK110.96 or SQK-RBK114.96), is ideal for multiplexing multiple samples to optimize costs while achieving high-quality genomic data. Below is a detailed overview:

Purpose and Applications

• Whole SARS-CoV-2 Genome Sequencing: Designed to tile the SARS-CoV-2 genome using overlapping amplicons for comprehensive genomic coverage.
• Multiplexing Samples: Enables pooling of multiple samples with rapid barcoding for cost-effective sequencing.
• Flexibility: Adaptable for various ONT sequencing platforms, such as MinION, GridION, and PromethION.

Key Features

1. Integrated Workflow:
   • Reverse transcription and PCR tiling with pre-designed primers.
   • Rapid barcoding to allow multiplexing and pooled sequencing.
   • Compatible with software tools like MinKNOW and Guppy for data deconvolution.
2. Tiling Primers:
   • Primer sets based on the Primal Scheme by Freed et al., 2020.
   • Updated to include additional primer 28 for enhanced Omicron variant coverage.
   • Regular updates ensure compatibility with emerging SARS-CoV-2 mutations.
3. Compatibility:
   • Works with Rapid Barcoding Kits (SQK-RBK110.96 and SQK-RBK114.96).
   • Suitable for various ONT flow cells and sequencing devices (e.g., FLO-MIN114, FLO-PRO114M).
4. Capacity:
   • Supports up to 576 RT/PCR reactions per kit.
   • Ideal for large-scale or routine surveillance projects.
5. Barcoding and Sample Pooling:
   • Supports Rapid Barcoding Kit for fast attachment of DNA barcodes.
   • Allows pooling and simultaneous sequencing of multiple samples.

Kit Contents

Workflow

1. Reverse Transcription:
   • SARS-CoV-2 RNA is reverse-transcribed using the LunaScript RT SuperMix.
2. PCR Amplification:
   • Tiled PCR generates overlapping amplicons using Midnight Primer Pools A and B.
3. Barcoding:
   • Barcodes are attached to DNA ends using a compatible Rapid Barcoding Kit.
4. Purification:
   • Pooled samples undergo SPRI purification before sequencing.
5. Sequencing:
   • Sequenced on ONT devices. Results processed using MinKNOW and Guppy.

Protocols

• PCR tiling of SARS-CoV-2 virus - Rapid Barcoding Kit 96 V14 and Midnight RT PCR Expansion (SQK-RBK114.96 and EXP-MRT001)
• PCR tiling of SARS-CoV-2 virus with rapid barcoding and Midnight RT PCR Expansion (SQK-RBK110.96 and EXP-MRT001)

Storage and Stability

• Shipped at 2–8°C.
• Long-term storage at -20°C.
• Shelf life: 3 months from delivery.

Third-Party Reagents and Equipment Required

• Reagents:
  • Nuclease-free water, ethanol (80%), and Qubit dsDNA HS Assay Kit.
  • PCR LoBind tubes and plates.
• Equipment:
  • PCR thermal cycler, magnetic separator, centrifuge, vortex, and Qubit fluorometer.
  • Multi-channel pipettes and other standard laboratory tools.

Advantages

1. Cost Efficiency:
   • Enables cost-effective sequencing through sample multiplexing.
2. High Accuracy:
   • Tiling PCR and optimized primers ensure robust coverage of the SARS-CoV-2 genome.
3. User-Friendly:
   • Pre-packaged reagents simplify the workflow, reducing the need for external suppliers.
4. Scalability:
   • Suitable for small and large-scale studies, with barcoding options to meet diverse needs.

Shipping and Logistics

• Environmentally friendly, temperature-controlled shipping.
• Worldwide delivery schedules tailored for efficient package handling.

The Midnight RT PCR Expansion Kit is a powerful tool for researchers and laboratories conducting SARS-CoV-2 surveillance, offering the flexibility, cost-effectiveness, and performance required for comprehensive genomic sequencing.