Difference between revisions of "Team:Toronto/Notebook-w14-tue"
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Revision as of 06:55, 19 October 2016
console.js:26
Tuesday, August 16
Tuesday, 8/16
Members Present: Alexander Sullivan, Karim Sarif, Zarifah Omar Ali, Celine Zhang, Marc Li, Cathy Cao
LAB:
Morning:
●
Test OD from previous day's overnight cell culture (taken from CCC)
A | B | C | |
1 | Time | Dilution | OD |
2 | ~1100 | None | 1.425 |
3 | ~1238 | None | 1.41 |
4 | 0.5 | 1.02 | |
5 | 0.33 | 0.799 | |
6 | ~1355 | None | 1.403 |
7 | 1.402 | ||
8 | 0.5 | 1 | |
9 | 1.014 | ||
10 | 0.3 | 0.746 | |
11 | 0.763 | ||
12 | 0.1 | 0.251 | |
13 | 0.267 | ||
Table1
We believe the current sample is going under the "Die-off" phase of bacterial growth as seen how the concentration of the none-diluted version.
NOTE: Previous days ( Monday, August 15) values were:
- After 1 hour passed: 0.011 (samples started at 3pm the past day)
- After 3.8 hours passed: 0.120
○
Due to believing the bacteria are in the die-off phase, we will need to make a new batch to grow and use
●
Checked the plates streaked yesterday
○
The CCC cells at OD 0.120 grew on Lb+Agar plates
■
This plate was used to grow four overnight culture with in liquid Lb and stored in the shaker in WB315 at 245 RPM in Falcon tubes
○
The CCC and CCF on Lb+Cam plates did not have any growth on them
Afternoon:
●
Miniprepped the following using NEB's Monarch (protocol has been added to the protocol folder) as well as nanodropped and ran on gel
A | B | C | D | E | |
1 | Sample name | ug/ul | 260/280 | 260/230 | ~Band size |
2 | C1 (white) | 33.7 | 1.36 | 0.69 | None |
3 | C1A | 77.6 | 1.87 | 2.12 | ~4kb, ~2kb |
4 | C1B | 52.7 | 1.85 | 2.05 | ~4kb |
5 | C1C | N/A | N/A | N/A | ~4kb, ~2,5kb |
6 | G1A | 67.8 | 1.89 | 2.2 | >10kb, ~8kb, ~4kb, ~2.5kb |
7 | G1B | 45.2 | 1.83 | 2.01 | ~4kb, ~2.5kb |
8 | G1C | 79.5 | 1.92 | 2.29 | >10kb, ~8kb, ~3.5kb, ~2kb |
9 | SP1A | 74.6 | 1.68 | 0.86 | >10kb, ~4kb, ~2.5kb |
10 | SP1B | 96.6 | 1.88 | 2.03 | >10kb, ~7kb, ~3.5kb, ~2.5kb |
11 | SP1C | 71.2 | 1.87 | 2.1 | ~3kb |
12 | SG1A | 52 | 1.92 | 1.97 | ~3kb |
13 | SG1B | 96.2 | 1.91 | 2.01 | >10kb, ~4kb, ~3kb, ~2kb |
14 | SG1C | 82.2 | 1.9 | 2.13 | ~3.5kb, ~2.5kb |
15 | R1A | 99.9 | 1.89 | 2.35 | >10kb, ~5kb, ~2.5kb |
16 | R1B | 96.3 | 1.94 | 2.25 | >10kb, ~5kb, ~2.5kb |
17 | R1C | N/A | N/A | N/A | None |
18 | P1A | 76.6 | 1.89 | 2.2 | >10kb, ~8kb, ~5kb, ~3.5kb |
19 | P1B | 120.2 | 1.73 | 1.19 | >10kb, ~7kb, ~4kb, ~3kb |
20 | P1C | 91.1 | 1.91 | 2.23 | ~6kb, ~3.5kb |
21 | Control Culture | 131.7 | 1.86 | 2.19 | ~10kb, ~5kb, ~4kb, |
22 | Red false positive | 140.3 | 1.91 | 2.34 | ~7kb, ~3-5kb |
23 | Contaminated cells | 60 | 1.83 | 1.96 | >10kb, ~7kb, ~2.5kb |
24 | Negative control | 5.4 | 1.87 | 0.34 | None |
Table2
The C1C is labbelled N/A for the nanodrop results due to a mislabelling causing us to miss nanodroppping the C1C. The RIC is labelled N/A for the nanodrop results due to the micro-centrifuge tube it was contained it somehow emptied itself. Also note that C1B gave an absorbance error the first time with 42.5, 2.00 and 30.07 for the first try and Na/N, Na/N and Na/N for the second time. The results you see is the third nanodrop. Nanodrop was done using 2ul of ellution buffer from the NEB Monarch Miniprep kit and 2ul of the miniprepped sample
○
Miniprepping:
■
Just some notes of things we did. We used 190ul of B1 (Plasmid Resuspension Buffer) instead of 200ul to make sure there was enough for 23 minipreps
■
The B3 (Plasmid Neutralization Buffer) is currently in the fridge in WB407, not the Monarch Miniprep box
■
Protocol reference: Monarch Miniprep Protocol
○
Gels:
■
Used 8ul of sample with 1.8ul of laoding dye and 6ul of NEB-2log DNA ladder at 100V for 60min
●
Weird things that happened:
○
Despite setting A at 11 and W at 300, the gel for some reason ran at 0.16A and 16W
○
Gel 2 TAE was hot after the run and a white thing appeared (see picture below)
○
Nanodrop results:
TO DO:
For the next day:
●
Measure OD of the overnight culture from streaked plate
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Create RbCl competent cells
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Miniprep one of the overnight cultures from streak plate and run a gel on it
○
Also run a gel on LacH and LacL
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Create more Lb+Agar and Lb+Cam plates
LAB MANAGERS: (For Zarifah Omar Ali)
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Autoclave tips, microcentrifuge tubes and 1.2L of MilliQ water
●
Inventory
