Plasmid DNA sequencing (SOP ID: 0002)

SOP ID:

0002

Revision nr.:

0.0

Release date:

November 8, 2019

Last release date:

November 8, 2019

Author(s):

Ada Serena Marletta

Approver:

Ada Serena Marletta

1. Scope

This Standard Operating Proceducere provides a step-by-step protocol for DNA plasmid sequencing with pre-mixed sequencing primers.

2. Background

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

3. Prerequisites

1,5 ml safe-lock tubes
0,2 ml strip tubes for PCR
96-wells plates for PCR
Sequencing Primers
Centrifuge
Sequencing labels (if required by DNA sequencing provider)
Biology grade water

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

Other (Oth): Third-party personnel and/or institution and/or authority, and/or service provider.

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	IF samples are to be submitted as single vials THEN go to step 2 ELSE go to step 8	Op
2	Prepare 2n vials, where n is the number of sequencing samples. Label n vials as {sample.name}_FW for sequencing w/ the forward primer Label n vials as {sample.name}_RV for sequencing w/ the reverse primer	Op
3	Add Forward sequencing primer (ID: XXXX) to all vials labeled as FW to the final concentration as specified by sequencing provider’s instructions	Op
4	Add Reverse sequencing primer (ID: XXXX) to all vials labeled as RV to the final concentration as specified by sequencing provider’s instructions	Op
5	Add to each vial the corresponding DNA plasmid to to the final concentration as specified by sequencing provider’s instructions	Op
6	Add to each vial mol. bio. grade water as much as it is needed to reach the sample total volume as specified by sequencing provider’s instructions.	Op
7	Submit samples to sequencing service provider according to its instructions	Op
8	For each 96 samples, prepare two 96-wells plates. Label one 96-wells plates FW for sequencing w/ the forward primer Label one 96-wells plates RV for sequencing w/ the reverse primer	Op
9	Add Forward sequencing primer (ID: XXXX) to each well of the 96-wells plate FW for sequencing w/ the forward primer to the final concentration as specified by sequencing provider’s instructions	Op
10	Add Reverse sequencing primer (ID: XXXX) to each well of the 96-wells plate RV for sequencing w/ the reverse primer to the final concentration as specified by sequencing provider’s instructions	Op
11	Add to each well of both 96-wells plates the corresponding DNA plasmid to the final concentration as specified by sequencing provider’s instructions	Op
12	Add to each well of both 96-wells plates mol. bio. grade water as much as it is needed to reach the sample total volume as specified by sequencing provider’s instructions.	Op
13	Submit plates to sequencing service provider according to its instructions	Op

8. Record Management

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

9. References

9.1 Standards

9.2 Other SOPs

The execution of this SOP does not reference to any other SOP

9.3 Reagents

No reference reagents are provided, Operator shall follow standard practice to procure required reagents

9.4 Sequences

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

9.5 Supplementary Documents

The execution of this SOP does not reference to any supplementary document

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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