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Oxford Nanopore TechnologiesFully scalable, real-time DNA/RNA sequencing technology
Oxford Nanopore DiagnosticsLamPORE – rapid, low-cost, scalable detection of SARS-CoV-2
London Calling 2022
18th - 20th May
PCR-cDNA Barcoding Kit
SQK-PCB111.24

Identification and quantification of full-length transcripts with highest output, with barcodes for up to 24 samples.

This is an Early Access product

This product has a 6 week lead time

$750.00
2Early Access
  • Access the latest products from Oxford Nanopore Technologies
  • Provide feedback into the innovation team as the product develops
  • Product Warranty may initially be limited to 1 month as it is so new
  • Product routinely iterates as new features and capabilities are added
  • Estimated lead time in store
  • Product subject to availability

Information

This kit is recommended for users who:

  • have a limited amount of input material
  • wish to multiplex samples to reduce price per sample
  • want to optimise their sequencing experiment for output
  • would like to identify and quantify full-length transcripts are interested in differential gene expression
  • want to characterise and quantify isoforms, splice variants and fusion transcripts

This is an Early Access product
For more information about our Early Access programmes, please see this article on product release phases.

The PCR-cDNA Barcoding Kit 24 features:

FeaturesProperties
Preparation time~160 mins + PCR
Input requirement4 ng poly(A)+ RNA, or 200 ng total RNA
RT RequiredYes
PCR requiredYes
Read lengthEnriched for full-length cDNA during PCR
Read type produced1D
Associated protocols- PCR-cDNA Barcoding Kit (SQK-PCB111.24)
Multiplexing optionsFlow Cell Wash Kit (EXP-WSH004)
Sequencing Auxiliary Vials (EXP-AUX110)
Pack size6 reactions
StabilityShipped 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

Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.

Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples: 

No barcodes6 barcodes12 barcodes24 barcodes
Flow cell price$500$500$500$500
Library price$99$99$99$99
Price per sample$599$104$54$28

The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 24 samples, from an input of as low as 4 ng polyadenylated RNA. Users who do not have polyadenylated enriched RNA can use 200 ng of total RNA but additional optimisation may be required.

The protocol uses a strand switching method to select for full length transcripts, allowing the identification of splice variants, with the incorporation of unique molecular identifiers (UMI) during this step. Taking full-length polyadenylated RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 24 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification using primers that contain 5’ tags and facilitate the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and the Rapid Sequencing Adapters are added to the pooled mix.

This cDNA kit (Kit 11) includes the Rapid Adapter T (RAP T), with two key features: Firstly, the adapter is higher capture, enabling lower flow cell loading amounts. Secondly, the adapter contains the fuel fix technology, enabling users to run long experiments without the need for fuel addition during the run.

The PCR-cDNA Sequencing Kit also includes a new cDNA RT adapter and RT primer to prime cDNA synthesis from the end of a transcript to reduce overlaps during the reverse transcription step and to allow users to measure polyA tail lengths.

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

Taking full-length polyadenylated RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 24 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification using primers that contain 5’ tags and facilitate the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.

PCS111

Protocols

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

What's in the box

The PCR-cDNA Barcoding Kit 24 contains 24 unique barcodes and enough reagents for 6 reactions:

SQK-PCB111.24 2

NameAcronymCap colourNo. of vialsFill volume per vial (μl)
Strand Switching Primer IISSPIIViolet1350
RT PrimerRTPYellow1200
cDNA RT AdapterCRTAAmber1200
Annealing BufferABOrange1200
Rapid Adapter TRAP TGreen110
RAP Dilution BufferRDBClear1100
Elution BufferEBBlack21,500
Short Fragment BufferSFBWhite47,500
Sequencing Buffer IISBIIRed1500
Loading Beads IILBIIPink1360
Loading SolutionLSWhite cap, pink label1400
Barcode Primers 1-24BP01-24White2410
Flush BufferFBBlue61,170
Flush TetherFLTWhite cap, purple label1200

Compatibility

The PCR-cDNA Barcoding Kit 24 can be used together with:

Kits

  • Flow Cell Wash Kit (EXP-WSH004)
  • Sequencing Auxiliary Vials (EXP-AUX002)

Flow cells

  • FLO-MIN106D
  • FLO-PRO002

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION