10  Introduction to the molecular exercise physiology lab

10.1 Health and safety in the lab

To ensure lab work is performed according to current rules and regulations regarding health and safety, the document «Retningslinjer Idrettslab» about this has been formed. The document describes standard operating procedures (SOP) pertinent to the molecular exercise physiology lab, as well as the “testlab”. All students and employees working in either of the labs are responsible to acquire knowledge about these SOPs. This document can be found here: Retningslinjer Idrettslab

In the molecular lab, the greatest health and safety hazards are the chemicals we handle. To assist us in safe chemical handling, we use the digital chemical database EcoOnline. Username: idrett, password: idrett. In this database, you can find information about all the chemicals we have in our lab. Each chemical has its own safety data sheet (SDS). For all chemicals you work with, you should read the SDS and work according to recommendations for protective equipment and handling of the chemical.

We have the following protective equipment in the molecular lab: lab coat, gloves, facial mask, eye protection. In addition we have two fume hoods where we can work with extra hazardous chemicals to avoid toxic fumes, vapours and dust. The fume hood also provides a physical barrier to chemical spills.

If you spill or spray any chemicals on yourself, somebody else or on the floor, benchtop etc., it’s important that you know how to handle this. Information about this can be found in the chemical’s SDS.

Another important aspect of health and safety in the lab is correct storage of chemicals. Chemicals that are either toxic, explosive, flammable, oxidizing, compressed gas, corrosive, irritant, environmental damaging and/or health hazards should be stored appropriately. Information about where a chemical should be stored can be found in the SDS. The lab manager is responsible for correct storage of chemicals, but all lab users are responsible for placing the chemical they’ve used back where it belongs.

All chemicals need to be handled appropriately. This means that its not allowed to throw all chemicals in the sink or regular trash bin. Some chemicals need to be declared by official waste handlers (GLØR). The waste of these chemicals should be stored in appropriate containers. The lab manager is responsible for making sure that such containers are available in the lab and that they are marked properly. Information about which chemicals that need to be declared can be found in the SDS, or by asking the lab manager. Generally, all biological waste (saliva, blood, muscle, urine) should be thrown in yellow waste bins, and nothing but biological waste should be thrown in yellow waste bins.

10.2 Good laboratory practice

• Standard work equipment in the lab is lab coat and gloves. No sandals with open toe.
• Never drink or eat, or keep food and drinks in the lab.
• Benchtops should be clean and tidy.
• Never work alone in the lab.
• Always come prepared: plan, acquire knowledge about the chemicals you will work with, make sure you have all the equipment and consumables you need.
• Work according to protocol and write down everything you do (elab). If you have to do something outside protocol, make sure to always write down what you did and why.
• ALWAYS mark the tubes/box you are working with (content, name/initials, date/year, project ID)
• Ask the lab manager if in doubt
• Try to keep the lab slightly tidy while working – others are also working there
• Precision/accuracy and focus are very important while working in the lab!

10.3 The laboratory journal

Your laboratory journal fills several purposes. It helps you in keeping track of what you have done, why you did it, what were the results of your experiments and what were your conclusions. The journal also helps your collaborators (fellow students, supervisors, laboratory managers) to understand what you have done. In a broader perspective the laboratory journal can be viewed as a primary source of data for your prospective manuscript, and when the manuscript has been published, the journal is source data kept for records at your department.

10.4 Keeping it digital - Using our electronic lab journal

After a couple of years running our lab in Lillehammer, we have arrived at the conclusion that we need to organize the molecular lab using a electronic laboratory journal system. This will solve several issues with organizing laboratory work like storage of data, collaborating, running multiple projects in parallel, having guests and students contributing to projects etc.

Traditionally, laboratory journals are stored in a fire safe cabinets in the lab, in notebooks. In some labs, each page must be “read and understood” and subsequently signed by a lab administrator or principle investigator. People working in laboratories typically have strong feeling about their laboratory journal system, regardless of digital or analog alternatives. The above observations indicate that, as a new lab member you should take care to learn the laboratory journal system and culture of the lab. This will make your work more important to the lab, as it will contribute to the collective knowledge based on the groups work.

Our electronic laboratory journal system can be found at elab.inn.no. As a student at inn you may log in using your student username and password. The system has link to the documentation of the software which can also be found through this web address: doc.elabftw.net/. As a student you will be added to the group related to this course.

10.4.1 What to write in the journal

A laboratory journal should consist of all necessary information needed to replicate your experiments. The tricky thing is that you do not always know what information is needed to do a replication. This means that you will need to include all relevant information about your experiment, and a bit more, within reasonable limits.

10.4.2 Structure of an entry

In the electronic journal (elab.inn.no), entries are recorded as experiments. An experiment starts on a specific date, it has a status, a title and tags can be added. This will make it easy to keep track of the entry. eLabFTW will keep a specific record of your entry and time stamp it. The actual entry has three parts, namely purpose, methods and results. These are suggested in the template when you start a new entry.

The purpose of an experiments should give a clear description of why you are performing the experiment. This description could be “To extract and analyze DNA for ACTN3 genotyping”. Further descriptions could be necessary if you are performing more complex experiments. Maybe you found something out in previous experiment that needs to be validated in this experiemnt. If you spend some time to write this up, your subsequent report will be easier to write and your collaborators will understand what you did.

The methods in an experiment “need to include all relevant information about your experiment, and a bit more, within reasonable limits”, as already mentioned above. A detailed protocol should be included. If you are using pre-written protocols, this entry should refer to this protocol and include any changes made to the protocol. You should also note batch numbers on chemicals and reagents etc. If an experiment fail, a complete record of reagents and machines etc. will make it easier to track potential methodological issues.

The method describes the experiment, e.g. by a step-by-step approach. This section includes recipes of solutions, what samples were used, in what order different steps of the protocol was done, if any problems occurred and so on. Remember to write explicit, you might remember tomorrow, but in one week or one year, you have no idea. A method section (as the other sections) can refer back in the laboratory journal system. For example, maybe you prepared and validated the lysis buffer in a previous experiment, write this and refer to the experiment describing the buffer. A part of a method section can look something like this:

Overview: Freeze dried muscle samples are homogenized in lysis buffer (Refer to experiment: Lysis buffer test) and protein concentrations are determined in the plate reader (raw data included below). Based on protein concentrations, supernatant is normalized to a common 2 μg/μl. Step-by-step: 1. Freeze dry muscle over-night (at least 20 h). 2. Dissect away fat, connective tissue and blood 3. Move ~2 mg to a new tube and add 80 μl of ice-cold lysis buffer per mg of tissue. 4. …”

Finally, the results should be a description of the actual results, and a description of what this means. If you would pick up your experiment later this will help you understand the results. This also applies to collaborations. In eLabFTW you can include raw data files. This will be the primary way of storing raw data from machines and instruments. Importantly, eLabFTW will not give you a way of structuring large amount of data. This step could instead be done outside eLabFTW.

10.4.3 Relationship between the laboratory journal and your samples, solutions, tubes etc.

When you do work in the laboratory, you will notice that you accumulate a lot of micro-centrifuge tubes, boxes full of intermediate sample preparations, solutions etc. To keep track of all this you need to “connect” the content of your laboratory journal entries to the place where you keep all your stuff. For example, when marking a new cryo-box, this should be done with information about what is the content of the box, the date when the box was “started”, your name, and the experiment. Your name, date and experiment can be tracked back to your laboratory journal entries. This means that when your collaborator finds a mystical “Sample X1 2017 10 10, experiment Y”, she can go back to your journal and find out what you did. In summary: label everything!

10.5 Protocols and experiments in the course

Protocols used in the course are as for now available at trainome.github.io.