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Midnight RT PCR Expansion

An expansion kit containing all the required reagents and primers to generate reverse-transcribed and PCR-amplified material from SARS-CoV-2 RNA samples.

SKU EXP-MRT001 Categorías ,
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Descripción

Information

This kit is recommended for users who:

  • Want to sequence the whole SARS-CoV-2 genome
  • Wish to multiplex samples to reduce price per sample

Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the «3rd Party Materials» tab for more detail.

The Midnight RT PCR Expansion (EXP-MRT001) is designed to be used with the Rapid Barcoding Kit 96 (SQK-RBK110.96) to provide all reagents needed for the complete workflow to sequence SARS-CoV-2 RNA samples.

The expansion kit provides all reagents and primers required to generate reverse-transcribed and PCR-amplified material from SARS-CoV-2 RNA samples, without the requirement of purchasing further components from multiple external suppliers. This expansion enables PCR tiling to generate overlapping amplicons across the whole SARS-CoV-2 genome before using the Rapid Barcoding Kit 96 (SQK-RBK110.96) to barcode and pool multiple samples together for sequencing.

The primers provided in this kit were initially designed by Freed et al., 2020, using the Primal Scheme. From the 1st February, we have included an additional primer 28 to address the drop out observed with the Omicron variant. As new mutations are identified in the SARS-CoV-2 lineages, we will continue to work closely with the community (including the entire ARTIC Nextwork, Nikki Freed, Olin Silander, Josh Quick, John Tyson, Nick Loman and many more) to rapidly respond and ensure performance of Oxford Nanopore Technologies’ protocols are always kept up-to-date, providing the best performance for robust SARS-CoV-2 whole genome sequencing.

Deconvolution of barcoded sequencing data is supported by MinKNOW and Guppy software, which classify the barcode sequencing and sort reads into corresponding folders.

Flow Cell Wash Kits can be purchased here for running fewer numbers of barcodes.

The Midnight RT PCR Expansion features:

Feature Property
Associated 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)

Pack size 576
Stability Shipped at 2–8°C

Long-term storage -20°C

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer

Shipping and logistics:

Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

Workflow

The Midnight RT PCR Expansion (EXP-MRT001) generates overlapping amplicons in a tiled fashion across the SARS-CoV-2 genome. First, the RNA samples are reverse-transcribed and then amplified by tiled PCR using separate primer pools. The primer pools are combined before moving into the rapid barcoding stage which attaches Rapid Barcodes to the DNA ends for samples to be pooled and SPRI purified.

What’s in the box

The Midnight RT PCR Expansion (EXP-MRT001) contains sufficient reagents for 576 RT/PCR reactions.

EXP-MRT001

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
LunaScript RT SuperMix LS RT Blue 3 500
Q5 HS Master Mix Q5 Orange 6 1,500
Midnight Primer Pool A MP A White 3 15
Midnight Primer Pool B MP B Clear 3 15

3rd party materials

Consumables

  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly-prepared 80% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with PCR seals
  • Qubit dsDNA HS Assay Kit (ThermoFisher, Q32850)
  • Qubit™ Assay Tubes (Q32856)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic separator, suitable for 1.5 ml Eppendorf tubes
  • Centrifuge capable of taking 96-well plates
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • Qubit fluorometer plate reader (or equivalent for QC check)
  • Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • Ice bucket with ice
  • Timer

Optional Equipment

  • Eppendorf 5424 centrifuge (or equivalent)
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • PCR-Cooler (Eppendorf)
  • Stepper pipette and tips

Compatibility

The Midnight RT PCR Expansion (EXP-MRT001) can be used together with:

Kits

  • Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
  • Rapid Barcoding Kit 96 (SQK-RBK110.96)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MIN114
  • FLO-PRO114M
  • FLO-MIN106
  • FLO-PRO002

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION

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