Team:Toronto/Notebook-w09-fri
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Friday July 15th
Friday, 7/15
Members Present: Kat, Hamed, Tam, Zarifah, Cathy, Alex
LAB:
Morning:
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PCR amplification of
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pSB1C3 backbone (2x one 50ul and one 20ul) labelled BB1A-20 and BB1B-50
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Short mCherry
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Long Linear GolS p118A
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Long Linear GolS
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Short Linear TetO-LacZ
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Restriction Enzyme digest of the aforementioned PCR amplified products
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PCR purification of the restriction enzyme digested products.
Afternoon:
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A gel was run on PCR products:
Lane 1: pSB1c3 bb 1A-20 , one band at ~2kb, faded band ~0.8kb
Lane 2: pSB1C3 bb 1B-50, one band at ~2kb
Lane 3: Short mCherry, single band at ~0.9kb
Lane 4: NEB 2-log DNA Ladder
Lane 5: Long linear GolS p118A, no bands
Lane 6: Long linear GolS, two faded bands at ~1kb and ~0.6kb
Lane 7: TetO-lacZ, no bands
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Wells contained 8ul of PCR sample with 1.6ul of DNA Loading Dye
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Well 4 was the DNA Ladder: 2-log DNA Ladder (0.1 - 10.0 kb)
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Gel ran at 100V for 60 min
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Visualize using GeneSnap with Transilliminator and Et/UV
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Nanodrop of the PCR amplified, RE digested and PCR purified products:
A | B | C | D | |
1 | SAMPLE | 260/280 | 260/230 | CONC (ng/ul) |
2 | 1a | 1.36 | 0.79 | 4.6 |
3 | 2.2 | 0.42 | 3.5 | |
4 | 2 | 1.76 | 0.73 | 9 |
5 | 1.49 | 1.06 | 8.7 | |
6 | 3 | 1.76 | 0.5 | 6.3 |
7 | 1.22 | 0.28 | 1.7 | |
8 | 1.18 | 0.71 | 5 | |
9 | 4 | 1.65 | 0.66 | 6.1 |
10 | 2.11 | 0.56 | 5 | |
11 | 5 | 0.89 | 1.72 | 1.2 |
12 | BB1 | 1.32 | 1.12 | 4.9 |
13 | 2.73 | 2 | 2.5 | |
14 | BB2 | 1.63 | 3.88 | 3 |
15 | 1.45 | 0.67 | 20.6 | |
16 | BB3 | 1.51 | 1.31 | 3.5 |
17 | BB4 | 1.16 | 2.95 | 1.7 |
18 | BB5 | 1.74 | 0.77 | 2.6 |
19 | ||||
Table1
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BB 1,2,3,4,5 were supposed to be combined together, but instead 1,2,3,4 were combined with sample 5. Therefore we restriction enzyme digested, PCR purified & nanodropped more BB (here distinguished as "pure"):
A | B | C | D | |
1 | SAMPLE | 260/280 | 260/230 | CONCENTRATION (NG/UL) |
2 | BB1 - pure | 1.46 | 0.67 | 8 |
3 | BB2 - pure | 1.62 | 0.66 | 2.4 |
Table2
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PCR Amplified and ran gel on the following:
Lane 1: pGolB - LacZ, no bands
Lane 2: Short TetO RcnR, no bands
Lane 3: Short PcrnA - LacZ, no bands
Lane 4: NEB 2-log DNA Ladder
Lane 5: Short TetO - GolS P118A, no bands
Lane 6: Short TetO - GolS, no bands
Lane 7: Negative control of Nuclease Free Water, no bands
A UV check was done on the gel and still only showed a band:
https://goo.gl/K08JRX
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GEL:
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Wells contained 8ul of PCR sample with 1.6ul of DNA Loading Dye
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Well 4 was the DNA Ladder: 2-log DNA Ladder (0.1 - 10.0 kb)
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Gel ran at 100V for 60 min
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Visualize using GeneSnap with Transilliminator and Et/UV
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PCR AMPLIFICATION:
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Please note an error was made where NEB's Phusion was used instead of ThermoFisher, this mistake could have led to the error you see here
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Also for some reason PCR reactions were >150ul for some unknown reason
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Ligation with samples 1-5 and with backbone.
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Transformation with ligated samples.
Administrative:
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Alex had discussion with P&P regarding obtaining a server from OG for the Syn Bio Network Platform
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P&P will have an email ready for sending on Monday
TO DO:
For the next day:
LAB TEAM:
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Check on and take pictures of transformed cells
LAB MANAGERS:
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Autoclave:
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More PCR tubes
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More micro-pipette tips
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600ml of MilliQ-water
