Oxford Nanopore Technologies SQK-RBK114 Rapid Barcoding kit protocol

Summary

This video tutorial demonstrates the process of rapid barcoding using the RBK114 kit. Lindsay, a researcher at UCL, walks through the essential steps, from preparing DNA samples to sequencing setup. Key steps include checking DNA quality, preparing barcodes, binding DNA to magnetic beads, washing, and eluting DNA. Additionally, Lindsay covers preparing the flow cell, loading the library, and ensuring everything is correctly processed for sequencing. The tutorial offers a comprehensive guide for accurate barcoding and sequencing setup, ensuring reproducible results in microbiological research.

Takeaways

• 😀 Ensure that genomic DNA is of high quality before starting the barcoding process to avoid poor sequencing results.
• 😀 Always measure the DNA concentration using a Cubit before starting the process and identify the DNA size.
• 😀 Using samples with varying DNA sizes (e.g., 60 kb vs 30 kb) can affect the efficiency of barcoding.
• 😀 DNA samples should be roughly the same size for even barcoding results, and different methods can be used to check the size (e.g., TapeStation, BioAnalyzer, gel electrophoresis).
• 😀 Prepare DNA samples with nuclease-free water to achieve a final volume of 10 µL, based on automatic calculations in the provided spreadsheet.
• 😀 Add rapid barcodes to the DNA samples, ensuring proper mixing and incubation at the specified temperatures (30°C for 2 minutes and 80°C for 2 minutes).
• 😀 After incubation, transfer the samples into low-binding tubes and add an equal volume of Ampure beads for DNA purification.
• 😀 Wash the DNA pellet with ethanol, carefully removing the ethanol without disturbing the magnetic beads.
• 😀 Dry the DNA pellet and add the elution buffer to release the DNA from the beads. Incubate at room temperature for 10 minutes before separating the beads again.
• 😀 For quantification, use the Cubit to measure 1 µL of the DNA sample and retain 11 µL for subsequent steps.
• 😀 Prepare the rapid adapter mix and adapter buffer in a separate tube, then add 1 µL of the mix to the DNA sample before preparing it for loading onto the flow cell.

Q & A

What is the RBK114 kit used for?

The RBK114 kit is used for rapid barcoding of samples in genomic DNA research, allowing for the efficient and accurate sequencing of multiple samples simultaneously.

How many samples can be processed with the RBK114 kit?

The RBK114 kit comes in two versions: one for processing 24 samples and another for 96 samples.

Why is it important to check the size of the genomic DNA before starting the experiment?

It is crucial to check the size of the genomic DNA because samples with different DNA sizes (e.g., 60 kb vs. 30 kb) do not barcode evenly, which could affect sequencing quality.

How can the size of genomic DNA be determined?

The size of genomic DNA can be determined using methods like the TapeStation, Bioanalyzer, or gel electrophoresis.

Why is it recommended to use high-quality, native DNA for sequencing?

Using high-quality, native DNA is important because sheared or damaged DNA does not sequence well, leading to poor results.

What is the role of the Ampure beads in the procedure?

The Ampure beads are used to purify the DNA after the barcoding step by binding to it and separating it from contaminants through magnetic separation.

How do you ensure that the ethanol washes the DNA pellet effectively?

To ensure proper ethanol washing, gently add ethanol to the tube while avoiding contact with the magnetic beads. This helps to wash away impurities without disturbing the bead-DNA complex.

What is the purpose of the incubation step with the elution buffer?

The incubation with the elution buffer allows the DNA to come off the beads, ensuring that the purified DNA is transferred into the elution buffer for subsequent steps.

How should you handle the elution after the DNA is separated from the beads?

After the DNA is separated from the beads, carefully remove the elution without disturbing the beads. Only a small amount is needed for cubit analysis, while the rest is saved for further steps.

What is the final step in preparing the DNA for sequencing?

The final step involves mixing the DNA library with sequencing buffer and library beads, then loading it onto the flow cell for sequencing. Proper handling of air bubbles during this step is essential to avoid interference with the sequencing process.