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PCR Barcoding Kit features
This kit is recommended for users who:
- wish to multiplex samples to reduce price per sample
- have a low starting amount of DNA
- want to optimise their sequencing experiment for throughput
- require control over read length
- are interested in targeted amplicon sequencing
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Introduction to the PCR Barcoding Kit protocol
This protocol describes how to carry out sequencing of low input genomic DNA using the PCR Barcoding Kit (SQK-PBK004).
- Please take note, from Batch 12 (SW04.10.0012) onwards of the PCR Barcoding Kit (SQK-PBK004), 1D Barcode Primer 01-12 (LWB 01-12) has changed name to Barcode Primer 01-12 (BP 01-12).
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 runLibrary preparation
You will need to:
- Fragment your DNA (this step is optional)
- Prepare the DNA ends for adapter attachment
- Attach PCR adapters supplied in the kit to the DNA ends, and amplify the fragments using barcoded primers
- Pool the barcoded samples, and attach rapid sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cellSequencing 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