Team:Toronto/Notebook-w14-tue

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Tuesday, August 16

Tuesday, 8/16
LAB:
Morning:
Test OD from previous day's overnight cell culture (taken from CCC)
A
B
C
1
TimeDilutionOD
2
~1100None1.425
3
~1238None1.41
4
0.51.02
5
0.330.799
6
~1355None1.403
7
1.402
8
0.51
9
1.014
10
0.30.746
11
0.763
12
0.10.251
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
IMG_20160816_124133.jpg
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Both plates looked like this. The one shown in this picture was used to later grow overnight-cultures from
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/ul260/280260/230~Band size
2
C1 (white)33.71.360.69None
3
C1A77.61.872.12~4kb, ~2kb
4
C1B52.71.852.05~4kb
5
C1CN/AN/AN/A~4kb, ~2,5kb
6
G1A67.81.892.2>10kb, ~8kb, ~4kb, ~2.5kb
7
G1B45.21.832.01~4kb, ~2.5kb
8
G1C79.51.922.29>10kb, ~8kb, ~3.5kb, ~2kb
9
SP1A74.61.680.86>10kb, ~4kb, ~2.5kb
10
SP1B96.61.882.03>10kb, ~7kb, ~3.5kb, ~2.5kb
11
SP1C71.21.872.1~3kb
12
SG1A521.921.97~3kb
13
SG1B96.21.912.01>10kb, ~4kb, ~3kb, ~2kb
14
SG1C82.21.92.13~3.5kb, ~2.5kb
15
R1A99.91.892.35>10kb, ~5kb, ~2.5kb
16
R1B96.31.942.25>10kb, ~5kb, ~2.5kb
17
R1CN/AN/AN/ANone
18
P1A76.61.892.2>10kb, ~8kb, ~5kb, ~3.5kb
19
P1B120.21.731.19>10kb, ~7kb, ~4kb, ~3kb
20
P1C91.11.912.23~6kb, ~3.5kb
21
Control Culture131.71.862.19~10kb, ~5kb, ~4kb,
22
Red false positive140.31.912.34~7kb, ~3-5kb
23
Contaminated cells601.831.96>10kb, ~7kb, ~2.5kb
24
Negative control 5.41.870.34None
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:
gel1.jpg
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gel2.jpg
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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)
IMG_20160816_222803.jpg
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If you have any idea of what this is or how it was formed, please say so because I am so confused what this even is
Nanodrop results:
C1White.TIF
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C1 (White)
CIB-take3.TIF
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C1B. The nanodrop result you are seeing is the third attempt as the first attemp gave an abosrbance error with the results of 42.5, 2.00 and 3.07. The second attempt game Na/N, Na/N and Na/N.
G1A.TIF
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G1A
G1B.jpg
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G1B
G1C.jpg
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G1C
SP1A.jpg
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SP1A
SPI-B.TIF
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SP1B
SPI-C.TIF
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SPIC
SGI-A.TIF
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SGIA
SG1B.jpg
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SGIB
SGI-C.TIF
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SGIC
R1A.jpg
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R1A
RI-B.TIF
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R1B
P1A.TIF
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P1A
P1B.jpg
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P1B
PIC.TIF
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PIC
ControlCulture.TIF
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Control Culture
RedFalsePositive.TIF
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Red False Positive
ContaminationCells.TIF
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Contaminated Cells
Negative control.jpg
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Negative Control
TO DO:
For the next day:
LAB TEAM: (For Alexander Sullivan, Cathy Cao, Hamed Tinafar, Tam Pham)
Measure OD of the overnight culture from streaked plate
Create RbCl competent cells
Miniprep one of the overnight cultures from streak plate and run a gel on it
Also run a gel on LacH and LacL
Create more Lb+Agar and Lb+Cam plates
LAB MANAGERS: (For Zarifah Omar Ali)
Autoclave tips, microcentrifuge tubes and 1.2L of MilliQ water
Inventory