Team:Toronto/Notebook-w09-mon

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Monday July 11th

Monday, 7/11
Members Present: Alex, Celine, Cathy, Kat, Bohdan, Hamed, Davesh (wiki), Andrea (wiki)
LAB:
Morning:
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Made more 2xYPTG
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Made 5ml overnight cultures of colonies from potential mCherry, GolS, and Gols P118A single colonies:
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Here are the plates from which we took the colonies. We had to leave them over the weekend so they're slightly dried up. Note that no additional colonies had popped up since Friday (and they were a little red on Friday too)
GolS.jpg
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"Ligated" GolS. GolS should not be red, therefore we suspect some potential RFP contamination from our positive control. Note 14 red colonies, 1 white colony. Overnight cultures were made of both the red and the white colony.
"Ligated" GolS P118A. Again, GolS is not red → possible contamination. 2 colonies. Overnight culture from one of the red colonies.
GolS P118A.jpg
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mCherry.jpg
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"Ligated" mCherry construct. 1 colony. mCherry should be red, however it is not. Overnight culture of single colony.
Afternoon:
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Ran PCR amplification with:
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TetO - GolS
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pgolB - LacZ
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pcrnA - LacZ
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TetO - GolS - P118A
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TetO - RcnR - C35A
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TetO - LacZ
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Gel results:
iGEM_July_11_2016_2020_ladder-annotated.png
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Lane 1: PCR TetO GolS, single band at ~0.6kb. Lane 2: PCR pgolB LacZ, faded single band at ~0.4kb. Lane 3: PCR pcrnA - LacZ at in between 0.4kb and 0.5kb. Lane 4: 2-log DNA Ladder (NEB's ladder size on left side of image, see reference 1 for full details). Lane 5: PCR TetO GolS P118A, single band found in between 0.6kb and 0.7kb. Lane 6: PCR TetO RcnR C35A, single band found in between 0.3kb and 0.4kb. Lane 7: PCR TetO LacZ, two bands at ~1kb and ~0.4kb. Lane 8: Nothing
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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
Administrative:
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Emailed Phil regarding printing
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Set up next meeting with OG (Wednesday)
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Set up Google Drive location for raw results:
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Viewable link - https://drive.google.com/folderview?id=0B6Z4m6vl-WRueEJnSjBUTXBWQTA&usp=sharing
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Editable link - Go to the iGEM Drive > Biomineralization > Results
Purchases made today:
A
B
C
D
E
F
1
NameSKUUnit PriceQuantityTotal (incl. tax)Status
2
TRYPTONE, BACTERIOLOGICAL, 1kgTRP402.159.4167.12Pickup 2-3 days.(EDIT- available 07/13/16
3
Hexagonal Weigh Boats Medium10770-45419.16243.3Available
Table1
TO DO:
For the next day:
LAB TEAM:
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Miniprep "Ligated" GolS, GolS P118A, mCherry constructs
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RE digest and ligation with mCherry and LacZ, using new T4 ligase and buffer (also using the ligation calculator)
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Transformation with ligated constructs.
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Make more LB Liquid Media and 1X TAE
Meetings/Notes:
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Weekly meeting slides to summarize Week 8 progress:
https://drive.google.com/folderview?id=0B6Z4m6vl-WRubVNmbUQtVmdtMEk&usp=sharing
References:
1.
https://www.neb.com/products/n3200-2-log-dna-ladder-01-100-kb