Team:UChicago/Protocols

Notebook > Protocols

Protocols page

General Lab Best Practices

Labeling

Label tubes at all time before storage. Label clearly with:

1. Contents in the tube
2. Date
3. Your name/Initials
4. Number

Notes on Sterile Techniques

• Turn the flame on before opening anything sterile.

• Aliquot everything you use to prevent contamination. Before pouring aliquots, flame the bottle rim quickly to keep sterile. Make new aliquots in new tubes with stock when running low.

• Unscrew but do not remove all caps to make it easier to work with in the moment.

• Flame the bottle rim quickly and then aliquot by pouring into 50ml conical tube. Never stick anything in LB bottle, always pour.

• Flame bottle rim again and close LB bottle.

• Take what you need from 50ml tube, keep sterile so it can be used repeatedly.

Lab Notebook Best Practices

Every lab notebook entry should contain…

1. Title: what technique you’re using, what you’re using it on (refer to specific tubes*)
2. Reference to protocol: are you using the standard protocol found in the masterlist?
3. Changes in protocol: have you done anything different from the standard protocol?
4. Conditions: did you run at 120V? for how long?
5. Mistakes: in case an experiment doesn’t work as a result of a mistake
6. Conclusion: was your experiment successful? why not? what’s next? what did you expect to see?
7. Your name: so others can ask questions to the right person. This is one reason why it’s critical to label tubes with the date, description, and your name

Best practices from Thomson’s How to Start–and Keep–a Laboratory Notebook: Policy and Practical Guidelines (see link and scroll down for examples):

“Although you may think you will remember what you did and why you did a certain experiment in a week’s time, YOU WILL NOT! And nor will anyone else in your laboratory. Hence the need for laboratory notebooks. In short, a laboratory notebooks is:

• a daily record of every experiment you do, think of doing, or plan to do
• a daily record of your thoughts about each experiment and the results thereof
• a record that would enable successive scientists, working on the same project, to pick up where you left off or reproduce your results”

“What goes into a laboratory notebook?

• a detailed account of every planned and executed experiment with the amount of detail that would enable a skilled scientist to determine what had been done, why it had been done, and what the results were
• dates accompanying every entry, account, or record
• [Links to outside resources used, formulas used for any calculations. -Alice]
• explanations of the significance of each experiment, as well as the observations, results and conclusions of the experiment
• details of each experiment (Remember, what may seem trivial or obvious at the time your experiment was conducted, may later be of critical importance.)
• personal comments (It is a living document, so stamp it with your own personality. Comments such as “SUCCESS AT LAST!! THIRD TIME LUCKY :)” are highly appropriate.
• photographs, computer generated data, and so forth should all be stuck into your notebook. a good photo matters!
• cross-references [For example, if you are starting a new experiment on 8/21 entry and are using the same protocol as already described in the Binder of Lab Practices, write on 8/21 entry, “following the protocol as described in the Binder of Lab Practices” -Alice]"

“A laboratory notebook is an important tool that goes well beyond research management, and keeping good records has implications for issues ranging from intellectual property management to the prevention of fraud.”

Steps to 3A Assembly & Labeling Guidelines

If from BioBrick in distribution kit plates (is an upstream or downstream part?):

1. resuspend plasmid DNA from kit plates
   • tube label e.g. lid: 18E plate 3, side: Pveg [date] [name]

▢ 2. transform plasmid DNA into DH5a cells (why those cells?) - plate label e.g. bottom of agar plate: [resistance] Pveg 18E3 [date] [name] [number if plating >1] ▢ 3. make overnight culture of a few isolated colonies - 15ml O.N. tube label e.g. side: [resistance] Pveg 18E3 [date] [name] [number if >1] ▢ 4. miniprep overnight culture

       - tube label e.g. lid: 18E3 mp [#], side: Pveg [date] [name] [concentration]ng/ul

▢ 5. nanodrop miniprep and write concentration on side of tube and in lab notebook, return 1ul to tube // if you have time, run 2ul on gel and based on band size, estimate concentration as well. ▢ 6. digest some of the miniprep

       - tube label e.g. lid: 18E3 dig [E, S] + [X, P], side: Pveg [date] [name]

▢ 7. run 2ul (why 2ul?) of the digest on a 1% gel, take photo and label ▢ 8. if digestion successful, then it’s ready to ligate. If you see label rubbing off, re-label it! Always use Sharpie pen.

Resuspending DNA from iGEM Kit Plates

Recipes

Making Agar Plates

Pouring the Plates

Making LB (500mL)

Agar Stab Protocols

Ligation Protocol

Transformation Procedures

Making Overnight Cultures

Miniprep Protocols

Making an agarose gel for gel purification

Enzymatic digestion

Gel extraction with the QIAGEN kit