Team:NDC-HighRiverAB/Notebook
NOTEBOOK
Wednesday, July 4, 2018
Today we made sterile LB and some new agar plates (5 with amp, 1 without + some extra plates that should have had amp but didn't.) Ampicillin was added at a ration of 1 uL per mL of agar liquid.
We autoclaved some waste and some pipette tips.
We received competent cells from fredsense to use for a transformation with our NDC 001-1 in the PET21a plasmid.
We had an issue with rehydrating the DNA. We added 15 uL of distilled water to 4 ug of plasmid, but after centrifuging and stirring it seemed like there was very little DNA solution, so we added an additional 10 uL of water before proceeding with the transformation. We let this sit for 7 minutes before moving forward with the transformation.
Because of needing to rehydrate the DNA with additional water, the competent cells were thawed on ice, distributed into tubes, and then put back on ice before the DNA was added.
We followed the transformation procedure from the Synthetic Biology Workshop Protocol Manual (University of Lethbridge 2016).
The following plates were made:
| Plate | Competent Cells | Transformed with Plasmid | Ampicillin |
|---|---|---|---|
| Control | X | ||
| Control | X | X | |
| Plate 1 | X | X | X |
| Plate 2 | X | X | X |
| Plate 3 | X | X | X |
| Plate 4 | X | X | X |
| Plate 5 | X | X | X |
Present: Mrs Côté, Ms Larocque, Emily D, Emily H, Daisy H, Miguel L, Micha B, Kristina C, Mya G
Thursday, July 5, 2018
2 colonies from each of Plates 1-4 (DH5a w/ NDCOO1-1 plasmid) were selected and placed in 5 mL LB with 5 uL ampicillin. These were left to incubate until tomorrow.
Present: Mrs. Cote, Mya G, Kristina C, Micha B
Friday, July 6, 2018
Ms Larocque did subcultures of all 8 overnight cultures - 5uL AMP, 5mL LB, 50uL cells to grow from 10am - 1pm.
Ran mini-prep, restritction digest, and gel on overnight culture plate 1A and 2A.
- Gel may have cooled too much before pouring
- We poked through gel wells on first two rows
We are testing our bacteria with the nitrophenyl solutions.
Robert's Emails about steps:
1. Take a sample of the overnight culture (100-500 uL) and pellet it in the centrifuge (max speed, 5 mins).
2. Add detergent to the phosphate buffer to get a 1% detergent phosphate solution.
3. Add nitrophenol at a 1:50 dilution so that we end up with a 1 mM nitrohpenol, 1% detergent phosphate solution (this is the reaction mixture).
4. Resuspend the cell pellet in the reaction mixture and incubate at either room temp or 37 degrees at 200 rpm.
5. Take a sample (250 uL) at the desired time points and centrifuge it to remove the bacteria. (NOTE: incubate on ice for 10 mins before centrifugation if you can as this will help stop the reaction from progressing while you centrifuge). Once pelleted, take the solution and store it in a clear vial in the fridge.
6. When all samples are ready, take a picture with a white background to see the progression of the reaction at each timepoint. If the tubes get in the way of taking a good picture, use a piece of parafilm with the paper backing removed and put a 10 uL drop on it. Emily can show you how to do this (we used to do this in iGEM to mix loading dyes into samples).
Emily Hicks made the solutions needed and these were our steps to fit in between (steps 3-6 above)
1. Make reaction micture 2.5mL of nitrophenyl mixture (2 of them - 50uL of nitro, 2450uL of detergent)
2. Pick 4 cultures (2 overnight 3A and 2B, 2 subcultures 2A and 2B) and spin down:
a) 500uL
b) 1000uL
3. Resuspend pellets in 300uL of reaction mixture
4. Incubate at 37*C
5. Check at 5 min = take 100uL out and put on ice for 10 min, centrifuge, check colour, take pictures)
6. Check at 10 min = take 100uL and repeat above
7. Check at 15 min
We didn’t end up removing the 100 uL samples as the reaction was happening very fast.
We didn't take an initial (0 min) picture for the nitrophenol oct - first picture was at 5 min.
Present: Emily Hicks (mentor), Emily H, Daisy H, Miguel, Micha, Mya, Mrs. Cote, and Ms. Larocque. (At various times throughout the lab).