Priming and loading the Flow Cell for nanopore sequencing (SOP ID:0006)

SOP ID:

0006

Revision nr.:

0.0

Release date:

November 26, 2019

Last release date:

November 26, 2019

Author(s):

Ada Serena Marletta

Approver:

Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for priming the flow cell and loading the cDNA library into it before running nanopore sequencing.

2. Background

This Standard Operating Procedure is intended for qualified personnel who was trained according to the ISO-9001 quality framework.

3. Prerequisites

Documents required:

MIAMI quality manual
SOP ID 0001

Material required:

Generic lab-, glass- and plastic-ware
MinION Mk1B (MIN-101B)
Flow Cell (FLO-MIN106D)
Direct cDNA Sequencing Kit (SQK-DCS109)
Flow Cell Priming Kit (EXP-FLP002)
1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, cat #AM9937)

4. Changes since last revision

No changes were made since last version

5. Role and responsibilities

Author (Au): Staff scientist properly trained to draft, review and disseminate standard operating procedures within the framework of ISO-9001

Approver (Ap): PI with the authority to approve, reject and withdraw standard operating procedures

MIAMI Quality Manager (QM): MIAMI authorized Quality Manager

Operator (Op): Staff scientist properly trained to execute the standard operating procedure

6. Monitoring Requirements

The present document can be reviewed at any time required by the author and the MIAMI Quality Manager

7. Procedure

Step	Action	Responsibility
1	Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at RT before placing the tubes on ice as soon as thawing is complete.	Op
2	Mix the Sequencing Buffer (SQB) and Flush Buffer (FB) tubes by vortexing, spin down and return to ice.	Op
3	Spin down the Flush Tether (FLT) tube, mix by pipetting, and return to ice.	Op
4	Open the lid of the nanopore sequencing device and slide the flow cell’s priming port cover clockwise so that the priming port is visible.	Op
5	After opening the priming port, check for small bubbles under the cover. Draw back a small volume to remove any bubble (a few µl) as follows: Set a P1000 pipette to 200 µl Insert the tip into the priming port Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip Care must be taken when drawing back buffer from the flow cell. The array of pores must be covered by buffer at all times. Removing more than 20-30 µl risks a loss of sequencing channels.	Op
6	Prepare the flow cell priming mix as follows: add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by pipetting up and down.	Op
7	Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 minutes.	Op
8	Thoroughly mix the contents of the Loading Beads (LB) by pipetting. Note that The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.	Op
9	In a new tube, prepare the library for loading as follows: 37.5 µl Sequencing Buffer (SQB) 25.5 µl Loading Beads (LB), mixed immediately before use 12 µl cDNA library prepared as described in SOP ID:0005 75 ul Total	Op
10	Complete the flow cell priming: Gently lift the SpotON sample port cover to make the SpotON sample port accessible. Load 200 µl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.	Op
11	Mix the prepared library gently by pipetting up and down just prior to loading.	Op
12	Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.	Op
13	Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION lid.	Op

8. Record Management

When completed and approved, the SOP will be published on MIAMI website

9. References

9.1 Standards

ISO-9001:2015
International System of Units

9.2 Other SOPs

The execution of this SOP refer to cDNA library preparation for nanopore sequencing (SOP ID: 0005)

9.3 Reagents

For reference reagents, please refer to the Prerequisites section.

9.4 Sequences

The execution of this SOP does not refer to any reference sequence

9.5 Supplementary Documents

This SOP is an adaptation of an example protocol using Direct cDNA sequencing kit available on Nanopore’s website.

10. Definitions

Measurements units are defined according to International System of Units

Metric prefixes are defined according to Metric (SI) Prefixes

Whenever possible, biological construct are defined according to the SBOL standard

Whenever possible, biological experiments are defined according to the metrology standards

11. Disclaimers

This Standard Operating Procedure (SOP) was developed based on a compilation of best available information, knowledge, experience, and general industry practices to provide guidance to MIAMI staff in performing the activities defined herein, in a consistent and standardized manner. This document does not contain regulatory or statutory requirements unless specified. The MIAMI consortium has made every attempt to present the information in a clear and concise manner for a variety of users. However, the MIAMI consortium is not responsible for the misuse or misinterpretation of the information presented herein. Under no circumstances shall The MIAMI consortium be liable for any actions taken or omissions made by non-MIAMI users of this document. In general, this document should be used as a reference. Differences may exist between the procedures referenced in this document and what is appropriate under project-specific conditions. This document does not represent an endorsement of practitioners or products mentioned in the document.

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