Difference between revisions of "Team:RHIT/Notebook"
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<li>NaCl</li> | <li>NaCl</li> | ||
<li>pH meter, neutral (pH 7) standard</li> | <li>pH meter, neutral (pH 7) standard</li> | ||
| − | <li>2M NaOH | + | <li>2M NaOH</li> |
<li>Dropper/ Pipette</li> | <li>Dropper/ Pipette</li> | ||
<li>Antibiotic (Ampicillin/ Cloramphenicol)</li> | <li>Antibiotic (Ampicillin/ Cloramphenicol)</li> | ||
Revision as of 16:40, 18 July 2018
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Toggle through the tabs and click on the dates or protocols below to learn more!
Lab Notebook
Week 1
6/6/18
Our first day in lab! After some training earlier in the week, we began our work in the actual lab. We learned how to make different broths and agars, and made Luria broth (LB) plates with chloramphenicol and LB plates with ampicillin.
Members: Ariel, Brittany, Emilie, Liz, Lining, Kaylee, and Elisa
Members: Ariel, Brittany, Emilie, Liz, Lining, Kaylee, and Elisa
6/7/18
Whatever we did this day
Week 2
Week 3
Week 4
Week 5
Week 6
7/12/18
We rehydrated our primers.
Members: Ariel and Elisa
Members: Ariel and Elisa
7/13/18
We calibrated the plate reader using serial dilutions of fluorescein for InterLab. The table below represents the plate wells and all values are measured in μM.
Members: Brittany
Members: Brittany
Week 7
7/16/18
We diluted primers for plasmid 1 to 100x and used those primers to amplify our plasmid 1 parts. We also made more chloramphenicol plates, transformed the plasmids for InterLab into competent NEB5-alpha cells, replated cells for InterLab onto chloramphenicol plates, and created a glycerol stock for our InterLab cells.
Members: Brittany and Elisa
Members: Brittany and Elisa
7/17/18
We inoculated cultures of transformed InterLab cells. We performed the InterLab Absorbance Calibration measured at 630 nm. We also performed the InterLab microsphere calibration measured at 630 nm. Finally, we transformed the part from well L4 into NEB5-alpha cells.
Members: Brittany and Liz
We ran parts 1-4 and 1-5 in a 2% gel and extracted part 1-4 (1-5 was not amplified.) We also designed new primers for the extraction and submission of parts that will allow us to put our parts in the pSB1C3 backbone for submission to the registry.
Members: Elisa
Members: Brittany and Liz
We ran parts 1-4 and 1-5 in a 2% gel and extracted part 1-4 (1-5 was not amplified.) We also designed new primers for the extraction and submission of parts that will allow us to put our parts in the pSB1C3 backbone for submission to the registry.
Members: Elisa
Week 8
Week 9
Week 10
Week 11
Protocols
Making LB Plates
This is the protocol for making LB gels. The measurements given can make approximately 24 plates. If fewer plates are wanted, the measurements can be halved to make approximately 10-12 gels.
Check the water level in the autoclave and put autoclave tape on the bottle. Ensure the lid is on but not tightened. Put the bottle in the autoclave and tighten the door. Set it for the liquid cycle and wait until the cycle is over and the pressure reaches 0 to open the door.
Put the bottle back on the stir plate and turn it to a low setting to prevent bubbles forming. Let the mixture cool until you can touch the bottle for several seconds comfortably. Add 600 µL of the chosen antibiotic (at 50mg/mL) to the bottle. Label all plates before you begin to pour. Wearing a glove, use the aseptic technique to pour the media into the plates and leave them to solidify.
Materials
- Large bottle with a screw-on lid
- Stir bar & Stir plate
- DI H2O
- Tryptone
- Yeast Extract
- Agarose
- NaCl
- pH meter, neutral (pH 7) standard
- 2M NaOH
- Dropper/ Pipette
- Antibiotic (Ampicillin/ Cloramphenicol)
- Sterile plates
Procedure
Fill a large bottle with lid with 570 mL of DI H2O. Insert a stir bar and place the bottle on the stir plate. Mix in 6g Tryptone, 3g yeast extract, 9g agar, and 6g NaCl. Ensure the solution has been mixed thoroughly and standardize the pH meter with the neutral standard solution. Add 2M NaOH to the solution dropwise until the pH reaches 7.Check the water level in the autoclave and put autoclave tape on the bottle. Ensure the lid is on but not tightened. Put the bottle in the autoclave and tighten the door. Set it for the liquid cycle and wait until the cycle is over and the pressure reaches 0 to open the door.
Put the bottle back on the stir plate and turn it to a low setting to prevent bubbles forming. Let the mixture cool until you can touch the bottle for several seconds comfortably. Add 600 µL of the chosen antibiotic (at 50mg/mL) to the bottle. Label all plates before you begin to pour. Wearing a glove, use the aseptic technique to pour the media into the plates and leave them to solidify.
Events
Midwestern Meetup - 6/30/18
On June 30th, two members of our team travelled to Michigan State University for the Midwestern iGEM Meetup. We spent the morning on a tour of the university’s botanical gardens learning about a range of plants and the history behind the gardens. After lunch, each team gave a short presentation and answered questions about their project. Then, we went on a tour of the Michigan State University iGEM team’s lab and facilities. Through this meetup, we made connections with other teams and have already started collaborating with the Michigan State team.
Ampacet Visit - 7/3/18
On July 3rd, two members of our team spoke with a representative from Ampacet in Terre Haute about our project. As a plastics expert, Dr. Jared Tatum explained the processes that are done at Ampacet. While discussing our project, Dr. Tatum offered the team PET plastic samples for our experiments. He will be giving us pure PET bottles, PET pellets, and PET powder. After explaining our experimental protocol ideas, Dr. Tatum advised us to change our procedures. Our original idea had been to let the bacteria eat the pellets as their only carbon source and observe the change. However, Dr. Tatum advised that we use the powder instead of the pellets because of the greater surface area to volume ratio. Additionally, he informed the team about the effect that humidity plays on the characteristics of pure PET. He encouraged us to monitor the humidity during our experiments and note any change in results based on a difference in humidity. Because pure PET is so susceptible to changes based on humidity, PET used in disposable water bottles contain a great amount of additives to stabilize the plastic and ensure long shelf life. Based on this new information, we began designing an experiment that would determine the effects of these additives on the ability of the bacteria to breakdown the PET.
Skype Call with Michigan State University iGEM - 7/9/18
On July 9th, the modeling and lab section of our team held a Skype conference with the Michigan State University team. During the Midwest Meetup, they showed interest in collaborating with us on lab and modeling aspects. We learned from the Skype meeting that they needed help creating a mathematical model and protein visualization model for their active site mutagenesis of ACC Deaminase. As our team had skills in creating mathematical models, we offered to help them create a system of differential equations to describe their system’s kinetics and genetics. The protein visualization was not something that we were very familiar with, so they decided to pursue other venues for help on that. In return, they offered to run a couple of our experiments involving their scanning electron microscope, as the one in our lab is not as sophisticated. After the call, they followed up with research articles with information we requested as necessary for building the model. We hope to have another call to discuss our results and coordinate our needs for the scanning electron microscope.