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 [http://www.iphandbook.org/handbook/chPDFs/ch08/ipHandbook-Ch%2008%2002%20Thomson%20Laboratory%20Notebooks.pdf 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]

1. make overnight culture of a few isolated colonies
   • 15ml O.N. tube label e.g.

side: [resistance] Pveg 18E3 [date] [name] [number if >1]

1. miniprep overnight culture
   • tube label e.g. lid: 18E3 mp [#], side: Pveg [date] [name] [concentration]ng/ul
2. 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.
3. digest some of the miniprep
   • tube label e.g. lid: 18E3 dig [E, S] + [X, P], side: Pveg [date] [name]
4. run 2ul (why 2ul?) of the digest on a 1% gel, take photo and label
5. 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

Protocol for resuspending DNA from the iGEM kit plates:

1. Poke through the aluminum foil on the well from which you want to take DNA with a 10ul tip with 10ul of nuclease-free water. Don’t take the foil off, it might cause cross-contamination between the wells.
2. Add 10 µL of dH2O. The water will turn red. Pipette in and out a few times to mix. Leave for 5 mins to make sure all the DNA dissolves.
3. DNA is ready to use for transformation. Use 2 µL for transformation. Acc. to the iGEM website, the concentration is high enough to use for transformation.

Recipes

Making Agar Plates

for 12 plates of 2 different antibiotics

Weight out:

• 5.0 g tryptone
• 2.5 g yeast extract
• 5.0 g NaCl
• 7.5 g agar

1. Add 250 mL of dH2O to a graduated cylinder.
2. Mix powder well to bring into solution.
3. Add dH2O to total volume of 500 mL and transfer to 1 L flask. All powder except agar should be dissolved.
4. Put on stirring hot plate and heat to boil for 1 min while stirring.
5. Autoclave at liquid setting for 15 minutes in a basin with some water making sure to loosen top.
6. Let cool to point you can pick up with gloves comfortably and split between 2 500ml jars, each containing 250ml LB-agar with flame on.
7. Let agar cool to ~55C (you should be able to pick up the jar without a glove) and add antibiotics

Pouring the Plates

1. Remove sterile Petri dishes from plastic bag (save the bag for storage)
2. Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not lift the cover off excessively (you should be able to just open up enough to pour).
3. Swirl plate in a circular motion to distribute agar on bottom completely.
4. Let each plate cool until its solid (~20 minutes) then flip so as to avoid condensation on the agar.
5. Store plates in plastic bags in fridge with: date and contents (note any additive).

Making LB (500mL)

Weight out:

• 5g tryptone
• 5g NaCl
• 2.5g yeast extract

1. Pour 250mL dH2O into 1L bottle.
2. Add ingredients and shake to mix
3. Pour 250mL dH2O to make total of 500mL
4. Autoclave on liquid setting

Agar Stab Protocols

1. Incubate agar stab from iGEM HG
2. With gloved hand, take sterile P1000 pipette tip and dip in the deep agar stab.
3. Streak on plate.
4. Streak again using another sterile pipette tip to spread bacteria over the plate.

Ligation Protocol

from [http://www.addgene.org/plasmid_protocols/DNA_ligation/ AddGene]:

1. Add amounts from highest to lowest (so water first). Flick to mix and pulse spin for 3 seconds.
2. Incubate at room temperature overnight.
3. If you’re not going to transform right away, heat inactivate tube at 65C for 10min.

Make controls if you can:

Transformation Procedures

TOP10 heat shock transformation procedure:

1. Let the cells thaw on ice.
2. Add your plasmid to the competent cells. Flick gently using index finger alone to mix. Do NOT vortex cells.
3. Leave on ice for 20 mins.
4. Place on the 42ºC heat block for 1 minute to heat shock.
5. Immediately place on ice for 2 minutes.
6. Add 1 mL 2X YT or SOC media.
7. Incubate in the 37ºC shaker for 1 hour. (We have no shaker, so we left it in a heat plate).
8. Centrifuge at 8 rcf for 30 seconds. Decant all but ~100 µL supernatant and resuspend.
9. Plate onto chloramphenicol (or antibiotic of choice) plates and use a sterile P1000 pipette tip to gently spread by hand while wearing gloves (no pipetman). Do not heat pipette tip. It’s okay if the tip leaves marks on the agar, but be as gentle as possible.

Ivan’s protocol for his competent cells in PCR tubes:

1. Let the cells thaw on ice.
2. Add your plasmid (~2ul) to the competent cells.
3. Leave on ice for 10 mins. [30min if you’re doing a ligation]
4. Heat shock at 42 degrees for 45 seconds

   a. Set the thermal cycler (PCR Machine) to 42 degrees

   b. Carry tubes in ice bucket to thermal cycler.

   c. Place tube inside for 45 seconds to heat shock.

5. Immediately put on ice for 2 minutes.
6. Add 1 mL SOC (or LB) media.
7. Incubate in the 37ºC shaker for 1 hour.
8. Centrifuge at 8 rpm for 30 seconds (pulse spin). Decant all but ~100 µL supernatant and resuspend.
9. Plate onto antibiotic of choice plates and use sterile, disposable plastic L-spreader to spread. Do not push on agar, just gently skim surface, it shouldn’t leave marks. If no spreaders are available, use a sterile P1000 pipette tip to gently spread by hand while wearing gloves (no pipetman). Do not heat pipette tip. It’s okay if the tip leaves marks on the agar, but be as gentle as possible.

Making Overnight Cultures

Miniprep Protocols

Making an agarose gel for gel purification

Enzymatic digestion

Gel extraction with the QIAGEN kit