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Introduction to the Ligation Sequencing Kit V14 (SQK-LSK114) protocol
This protocol describes how to carry out sequencing of amplicon sample using the Ligation Sequencing Kit V14 (SQK-LSK114). It is recommended that a Lambda control experiment is completed first to become familiar with the technology.
For information about Kit 14 chemistry, please see the Kit 14 sequencing and duplex basecalling info sheet.
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
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 End-prep Prepare the DNA ends for adapter attachment. 35 minutes 4°C overnight Adapter ligation and clean-up Attach sequencing adapters to the DNA ends and perform a bead clean-up. 30 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell.
-80°C for single-use long-term storage.
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 and convert it into basecalled reads