Sampling procedure for yeast culture (SOP ID: 0012)

SOP ID:

0012

Revision nr.:

0.0

Release date:

November 19, 2019

Last release date:

February 3, 2020

Author(s):

Ada Serena Marletta

Approver:

Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for sampling yeast culture during its growth in a bench bioreactor. The sampling procedure mainly depends upon the method employed to grow yeast, and sampling volumes may vary depending on the type of measurement to be performed downstream. Drafting of this Procedure is in progress.

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
MicroClave Connectors
Syringe w/Spinning Spiros (3 ml, 5ml, 10 ml)
Laminar Flow Hood
Ethanol
Spectrophotometer
Plastic cuvettes

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	IF you want to sample the culture medium before the addition of the inoculum, THEN go to step 2 IF you want to sample yeast culture during its growth (SOP ID:0010), then go to step 8	Op
2	Open a new sterile connector that will be referred to as “free connector”	Op
3	Open a new 10 ml Syringe w/Spinning Spiros and screw it to the connector attached to the bioreactor	Op
4	Collect 10 mL, unscrew the syringe and screw it to the free connector	Op
5	Switch on the laminar flow hood and, once the flow is stable, transfer the collected sample in a sterile tube. Take 1 ml for the OD600 blank measurement. Store the residual volume	Op
6	Swipe the connector attached to the bioreactor with a towel wetted with some ethanol, then remove the volume blocked in the collection pipe by pipetting 3 mL of air from the syringe connected to the manual air inlet	Op
7	Go to step 21	Op
8	Open a new 10 ml Syringe w/Spinning Spiros and screw it to the connector attached to the bioreactor	Op
9	Collect 500 μL of the culture to discard any possible liquid left in the pipe	Op
10	Open a new sterile connector that will be referred to as “free connector”	Op
11	Unscrew the syringe from the connector attached to the bioreactor, connect it to the free connector, and discard the volume.	Op
12	Unscrew the syringe from the free connector and screw it to the bioreactor	Op
13	Collect 5 mL, unscrew the syringe and screw it to the free connector	Op
14	Swipe the connector attached to the bioreactor with a towel wetted with some ethanol, then remove the volume blocked in the collection pipe by pipetting 3 mL of air from the syringe connected to the manual air inlet	Op
15	Switch on the laminar flow hood and, once the flow is stable, transfer sample collected at step 5 in a sterile tube	Op
16	Take 1 ml of the transferred sample, and measure OD600 in triplicate with disposable plastic cuvettes	Op
17	Calculate the sample volume required to have 1,5 x 10⁷cells with the formula V(ml)=(½)(1/OD_600mean), where OD_600mean is the value obtained from the average of the 3 replicates in step 8 (the formula is calculated according to the assumption that cell concentration for yeast culture with OD600=1 is 3 x 10⁷cells/ml)	Op
18	Transfer the volume containing 1,5 x 10⁷cells in the appropriate tubes chilled on ice and centrifuge the tubes at max speed for 2 minutes. For volumes < 500 ul, add sterile SC medium up to 1 ml final volume before centrifuging.	Op
19	Discard the supernatant with the help of a micropipette and store the pellets at -80 °C until required for total RNA extraction (SOP ID:0013).	Op
20	Repeat steps 8-19 at any required time point. Choose the most appropriate syringe with non-return valve according to the expected OD600 value at each time point.	Op
21	End of procedure	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

Yeast growth protocol in a bench bioreactor (SOP ID: 0010)

Total RNA extraction from yeast cells (SOP ID: 0013)

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

There are no supplementary documents.

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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