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PCR-cDNA Barcoding Kit

SQK-PCB109

$650.00

Identification and quantification of full-length transcripts with highest throughput, with barcoding for ≤12 samples

Includes a Flow Cell Priming Kit

  • Information

    This kit is recommended for users who:

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

    The PCR-cDNA Barcoding Kit features:

    Features

    PCR-cDNA Barcoding Kit

    Preparation time

    165 mins

    Input requirement

    1 ng poly-A+ RNA, or 50 ng total RNA

    RT Required

    Yes

    PCR required

    Yes

    Read length

    Enriched for full-length cDNA during PCR

    Read type produced

    1D

    Typical throughput

     •••

    Typical number of reads (on FLO-MIN106 at an average read length of 1 kb)

    7-12+ million full-length mapped reads per flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION

    Associated protocols

    PCR-cDNA Barcoding Kit

    Flow Cell Wash Kit

    Pack size

    6 reactions

    Storage

    Shipped at -20° C

    Long term storage -20° C

    Description

    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.

    The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 12 samples, from an input of as low as 1 ng poly-A+ RNA. Users who do not have poly-A+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required.

    Taking full-length poly-ARNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates 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.

     

    Shipping and logistics

    Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8° C, and -20° C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

    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.

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

  • Workflow

    Taking full-length poly-ARNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates 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.

     

     

    Data analysis

    Data from a PCR-cDNA sequencing experiment can be analysed using a number of bioinformatics tools and pipelines:

    • DESeq2 for differential gene expression profiling
    • pinfish for annotation of gene transcripts and novel gene isoform discovery
    • pipeline-transcriptome-de for performing differential transcript usage (DTU) and differential gene expression (DGE) analyses

    Oxford Nanopore Technologies offers easy-to-follow bioinformatics tutorials, which take the user step-by-step through each analysis pipeline. These tutorials are available in the Nanopore Community.

  • Protocols
  • Safety and legal
  • What's in the box

    The PCR-cDNA Barcoding Kit contains sufficient reagents to generate six sequencing libraries.

    What's in the box

     

    The Flow Cell Priming Expansion Pack is also supplied.

    EXP-FLP002

  • 3rd party materials

    Consumables

    • Agencourt AMPure XP beads
    • 1.5 ml Eppendorf DNA LoBind tubes
    • 0.2 ml thin-walled PCR tubes
    • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
    • Freshly prepared 70% ethanol in nuclease-free water
    • 10 mM dNTP solution (e.g. NEB N0447)
    • LongAmp Taq 2X Master Mix (e.g. NEB M0287)
    • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
    • RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
    • Exonuclease I (NEB, M0293)

    Equipment

    • Hula mixer (gentle rotator mixer)
    • Magnetic separator, suitable for 1.5 ml Eppendorf tubes
    • Microfuge
    • Vortex mixer
    • Thermal cycler
    • P1000 pipette and tips
    • P200 pipette and tips
    • P100 pipette and tips
    • P20 pipette and tips
    • P10 pipette and tips
    • P2 pipette and tips
    • Ice bucket with ice
    • Timer
    • Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf 022510509)
    • Qubit fluorometer (or equivalent for QC check)
  • Compatibility

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

    Kits

    • Flow Cell Wash Kit (EXP-WSH003)

    Flow cells

    • FLO-MINSP6
    • FLO-MIN106D
    • FLO-PRO002

    Devices

    • MinION Mk 1B
    • MinION Mk 1C
    • GridION Mk1
    • PromethION
Information

This kit is recommended for users who:

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

The PCR-cDNA Barcoding Kit features:

Features

PCR-cDNA Barcoding Kit

Preparation time

165 mins

Input requirement

1 ng poly-A+ RNA, or 50 ng total RNA

RT Required

Yes

PCR required

Yes

Read length

Enriched for full-length cDNA during PCR

Read type produced

1D

Typical throughput

 •••

Typical number of reads (on FLO-MIN106 at an average read length of 1 kb)

7-12+ million full-length mapped reads per flow cell on MinION/GridION; 50+ million mapped reads per flow cell on PromethION

Associated protocols

PCR-cDNA Barcoding Kit

Flow Cell Wash Kit

Pack size

6 reactions

Storage

Shipped at -20° C

Long term storage -20° C

Description

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.

The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 12 samples, from an input of as low as 1 ng poly-A+ RNA. Users who do not have poly-A+ enriched RNA can use 50 ng of total RNA but additional optimisation may be required.

Taking full-length poly-ARNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates 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.

 

Shipping and logistics

Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8° C, and -20° C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

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.

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

Workflow

Taking full-length poly-ARNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates 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.

 

 

Data analysis

Data from a PCR-cDNA sequencing experiment can be analysed using a number of bioinformatics tools and pipelines:

  • DESeq2 for differential gene expression profiling
  • pinfish for annotation of gene transcripts and novel gene isoform discovery
  • pipeline-transcriptome-de for performing differential transcript usage (DTU) and differential gene expression (DGE) analyses

Oxford Nanopore Technologies offers easy-to-follow bioinformatics tutorials, which take the user step-by-step through each analysis pipeline. These tutorials are available in the Nanopore Community.

What's in the box

The PCR-cDNA Barcoding Kit contains sufficient reagents to generate six sequencing libraries.

What's in the box

 

The Flow Cell Priming Expansion Pack is also supplied.

EXP-FLP002

3rd party materials

Consumables

  • Agencourt AMPure XP beads
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 10 mM dNTP solution (e.g. NEB N0447)
  • LongAmp Taq 2X Master Mix (e.g. NEB M0287)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
  • RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
  • Exonuclease I (NEB, M0293)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic separator, suitable for 1.5 ml Eppendorf tubes
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
  • Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf 022510509)
  • Qubit fluorometer (or equivalent for QC check)
Compatibility

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

Kits

  • Flow Cell Wash Kit (EXP-WSH003)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D
  • FLO-PRO002

Devices

  • MinION Mk 1B
  • MinION Mk 1C
  • GridION Mk1
  • PromethION
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