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PCR Barcoding Expansion 1-12 features
This kit is recommended for users who:
- want to optimise their sequencing experiment for throughput
- require control over read length
- would like to utilise upstream processes such as size selection or whole genome amplification
- want to multiplex up to 12 different samples
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Introduction to the PCR barcoding protocol
This protocol describes how to carry out PCR barcoding of DNA using the Ligation Sequencing Kit 1D (SQK-LSK110) and the PCR Barcoding Expansion 1-12 (EXP-PBC001). It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
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:
- Prepare the DNA ends for adapter attachment
- Attach barcoding adapters supplied in the kit to the DNA ends
- Amplify each barcoded sample by PCR, then pool the samples together
- Repair the DNA, and prepare the DNA ends for adapter attachment
- Attach 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