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Rapid PCR Barcoding Kit 24 V14 features
This kit is recommended for users who:
- Wish to multiplex samples to reduce price per sample
- Have a low starting amount of DNA
- Require a simple library preparation procedure
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Introduction to the Rapid PCR Barcoding 24 V14 protocol
This protocol describes how to carry out rapid low input PCR barcoding of genomic DNA using the Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24). There are 24 unique barcodes, allowing the user to pool up to 24 different samples in one sequencing experiment.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA, and check its length, quantity and purity.
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
You will need to:
- Tagment your DNA using the Fragmentation Mix in the kit
- PCR using the barcoded primer supplied in the kit
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
Note that after the PCR, the average length of DNA fragments should be <5 kb.
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
- Start the EPI2ME software and select the barcoding workflow