Information
This kit is recommended for users who:
- Wish to multiplex up to 24 samples to reduce price per sample
- Have a low starting amount of gDNA
- Require a simple library preparation procedure
This is an Early Access product.
For more information about our Early Access programmes, please see this article on product release phases.
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.
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 barcodes | 6 barcodes | 12 barcodes | 24 barcodes |
|---|
| Flow cell price | $500 | $500 | $500 | $500 |
| Library price | $99 | $99 | $99 | $99 |
| Barcode price | - | $25 | $50 | $75 |
| Price per sample | $599 | $104 | $54 | $28 |
The Rapid PCR Barcoding Kit 24 V14 features
| Feature | Property |
|---|
| Preparation time | 15 minutes + PCR |
| Input requirement | 1-5 ng gDNA per sample |
| PCR Required | Yes |
| Fragmentation | Transposase-based |
| Read length | ~2 kb |
| Kit chemistry | Kit 14 (V14) |
| Associated protocols | Rapid sequencing DNA V14 - PCR Barcoding Kit 24 (SQK-RPB114.24) |
| Pack size | 6 reactions |
| 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. |
The Rapid PCR Barcoding Kit 24 V14 offers the fastest and simplest method of preparation of barcoded libraries for low quantities of gDNA (1-5 ng), with only ~15 minutes of hands-on preparation time.
This kit has been upgraded to use our Kit 14 chemistry, which includes improved modal raw read sequencing accuracies (Q20+) with high output on our latest nanopore: R10.4.1. The flow cell priming and sequencing reagents are also included as part of the sequencing kit and have been reformulated to be compatible with the Kit 14 chemistry upgrade and R10.4.1 nanopore. Other updates include higher capture rate of gDNA to enable lower flow cell loading amounts, and fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.
At the heart of the kit is a transposase which simultaneously cleaves template molecules in each sample and attaches tags, which contain primer binding sites, to the cleaved ends. The kit contains 24 primers which are then used to amplify each sample: each primer contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled, and Rapid Sequencing Adapters are added to the pooled mix.
Deconvolution of barcoded sequencing data is supported by the MinKNOW software, the stand-alone Guppy basecaller, and EPI2ME, all of which classify the barcode sequence and sort reads into corresponding folders.
Further considerations:
As well as being used to generate more material where starting material is limited, PCR can be used where sample purity is compromised, which can inhibit library preparation efficiency. PCR selects for molecules which have been successfully adapted, and generates more of them, diluting the original impurity. It is often observed that a sample which produces poor sequencing results can be improved by the inclusion of PCR.
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 Rapid PCR Barcoding Kit 24 V14 offers the fastest and simplest method of preparation of barcoded libraries for low quantities of gDNA (1-5 ng), with only ~15 minutes of hands-on preparation time.
At the heart of the kit is a transposase which simultaneously cleaves template molecules in each sample and attaches tags, which contain primer binding sites, to the cleaved ends. The kit contains 24 primers which are then used to amplify each sample: each primer contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled, and Rapid Sequencing Adapters are added to the pooled mix.
Protocols
To access the protocols, you will need to register for a Nanopore Community account.
What's in the box
The Rapid PCR Barcoding Kit 24 V14 contains enough reagents for six reactions.
| Name | Acronym | Number of vials | Cap colour | Fill volume per vial (µl) |
|---|
| Rapid Barcode Primers | RLB01-24 | 24 (one per barcode) | Clear | 15 |
| Fragmentation Mix | FRM | 1 | Brown | 160 |
| Rapid Adapter | RA | 1 | Green | 15 |
| Adapter Buffer | ADB | 1 | Clear | 100 |
| AMPure XP Beads | AXP | 3 | Amber | 1,200 |
| Elution Buffer | EB | 2 | Black | 500 |
| EDTA | EDTA | 1 | Blue | 700 |
| Sequencing Buffer | SB | 1 | Red | 700 |
| Library Beads | LIB | 1 | Pink | 600 |
| Library Solution | LIS | 1 | White cap, pink label | 600 |
| Flow Cell Flush | FCF | 1 | Clear cap, light blue label | 8,000 |
| Flow Cell Tether | FCT | 1 | Purple | 200 |
Note: This product contains AMPure XP Reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
3rd party materials
Consumables
- LongAmp Taq 2X Master Mix (e.g. NEB, M0287)
- (Optional) Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
- Ice bucket with ice
- Microfuge
- Timer
- Thermal cycler
- Magnetic rack
- Hula mixer (gentle rotator mixer)
- P1000 pipette with tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Multichannel pipette
- Qubit fluorometer (or equivalent for QC check)
Note: The above are 3rd party reagents required for the standard prep. For other workflows using this kit, extra 3rd party reagents may be required.