cDNA preparation for nanopore sequencing (SOP ID: 0003)

Revision nr.:
Release date:
November 19, 2019

Last release date:

November 29, 2019
Ada Serena Marletta


Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for cDNA synthesis starting from polyA mRNA. The synthesized cDNA is then subjected to Oxford 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
  • 100 ng PolyA+ RNA
  • Direct cDNA Sequencing Kit (SQK-DCS109)
  • Agencourt AMPure XP beads
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 10 mM dNTP solution (e.g. NEB N0447)
  • LongAmp Taq 2X Master Mix (e.g. NEB M0287)
  • Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
  • RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
  • RiboShredder (Epicentre, RS12500), or RNase Cocktail Enzyme Mix (ThermoFisher, AM2286)

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.
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