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Introduction to the Rapid Barcoding Kit V14 kits
This protocol describes how to carry out rapid barcoding of genomic DNA using the Rapid Barcoding Kit 24 and 96 V14 (SQK-RBK114.24 or SQK-RBK114.96). These kits use our most recent Kit 14 chemistry and are optimised for fast library preparation requiring minimal laboratory equipment.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA, and check its length, quantity and purity using the Input DNA/RNA QC protocol. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment and third-party reagents.
- Download the software for acquiring and analysing your data.
- Check your flow cell to ensure it has enough pores for a good sequencing run.
Library preparation
The table below is an overview of the steps required in the library preparation, including timings and stopping points.
Library preparation step Process Time Stop option DNA barcoding Tagmentation of the DNA using the Rapid Barcoding Kit V14 15 minutes 4°C overnight Sample pooling and clean-up Pooling of barcoded libraries and AMPure XP Bead clean-up 25 minutes 4°C overnight Adapter ligation Attach the sequencing adapters to the DNA ends 5 minutes We strongly recommend sequencing your library as soon as it is adapted Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes Sequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads.
- Demultiplex the reads by barcode using MinKNOW, the Guppy software, or the barcoding workflow in EPI2ME.