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Midnight RT PCR Expansion
EXP-MRT001

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

$2,880.00

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

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.

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.

Shipping and logistics:

Flow cells and kits are shipped together at 2–8°C.

Upon receipt, please place the right product in the right long-term storage location.

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.

Protocols

To access the protocol, you will need to register for a Nanopore Community account.

What's in the box

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

EXP-MRT001

NameAcronymCap colourNumber of vialsFill volume per vial (µl)
LunaScript RT SuperMixLS RTBlue3500
Q5 HS Master MixQ5Orange61,500
Midnight Primer Pool AMP AWhite315
Midnight Primer Pool BMP BClear315

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 (SQK-RBK110.96)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106
  • FLO-PRO002

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION