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Tuesday, August 16

Tuesday, 8/16

Members Present: Alexander Sullivan, Karim Sarif, Zarifah Omar Ali, Celine Zhang, Marc Li, Cathy Cao

LAB:

Morning:

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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

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Due to believing the bacteria are in the die-off phase, we will need to make a new batch to grow and use

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Checked the plates streaked yesterday

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The CCC cells at OD 0.120 grew on Lb+Agar plates

IMG_20160816_124133.jpg

Both plates looked like this. The one shown in this picture was used to later grow overnight-cultures from

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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

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The CCC and CCF on Lb+Cam plates did not have any growth on them

Afternoon:

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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

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Miniprepping:

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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

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The B3 (Plasmid Neutralization Buffer) is currently in the fridge in WB407, not the Monarch Miniprep box

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Protocol reference: Monarch Miniprep Protocol

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Gels:

gel1.jpg

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

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Weird things that happened:

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Despite setting A at 11 and W at 300, the gel for some reason ran at 0.16A and 16W

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Gel 2 TAE was hot after the run and a white thing appeared (see picture below)

IMG_20160816_222803.jpg

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

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Nanodrop results:

C1White.TIF

C1 (White)

CIB-take3.TIF

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.