Difference between revisions of "Team:Toronto/Notebook-w05-thu"

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<li><a href="https://2016.igem.org/Team:Toronto/Safety"><span>safety</span></a></li>
 
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Revision as of 05:53, 19 October 2016

Thursday June 16, 2016

Thursday, 6/16
TheseMembers Present: Andrea, Kat, Marc, Karim, Bohdan, Hamed, Davesh
LAB:
Morning:
- Verified that cells did not transform
2X - 5pg.jpg
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Transformation with 5pg RFP and CCA cells (above) and CCB cells (below). Note that no colonies grew
CCA And CCB cells transformed with 10pg of RFP. Note that nothing grew.
2X - 10pg.jpg
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2X - 20pg.jpg
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CCA and CCB cells transformed with 20pg of RFP. Note that nothing grew.
CCA and CCB cells transformed with 50pg RFP. Note three colonies on CCB plate.
2X - 50pg.jpg
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- Made 1ml aliquots of nuclease free water, stored on a rack on the shelf in 403
- Miniprepped RFP:
RFP_1.jpg
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5ml culture innocuated with a single colony in a 50ml falcon tube, kept O/N in a orbital shaker at 200 rpm.
Artisitic shot of RFP & the Toronto skyline
RFP_2.jpg
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Nanodrop from RFP, taken from the initial transformation with the chemically competent cells, batch B (CCB)
A
B
C
D
E
1
Sample:260/280:260/230:Concentration:
2
RFP - MP1A1.631.2716.7 ng/uL
3
RFP - MP1B1.430.6115.0 ng/uLused for transformation
4
RFP - MP2A1.590.618.2 ng/uL
5
RFP - MP2B1.550.6512.8 ng/uL
Table1
Eluted with nuclease free water, and blanked the nanodrop woth nuclease free water
Afternoon:
Obtained chemically competent cells from Kayla:
Followed the bacterial transformation protocol, set up the following transformations:
Note that we suspect that one of the primary reasons that the cells did not transform was the heat shocking time (60sec) was too high. We have lowered this to 45sec.
A
B
C
D
E
1
Competent Cell Batch ID:
2
CCACCBCC0
3
DNA added:0.5pg0.5pg50pg
4
10pg10pgRFP1B
5
50pg50pg
6
RFP1BRFP1B
7
Control - no plasmidControl - no plasmid
8
Table2
Legend: CCA (competent cells batch A), CCB (competent cells batch B), CC0 (Competent cells obtained from Kayla as a positive control)
Also streaked out RFP on LB + CAM
Administrative:
TO DO:
For the next day:
LAB TEAM:
- Prep & freeze the cells for s12
- Check on cell transformations
- If not worked -> New chemically competent cells
- -> Transformation protocol (thermomixer)
-> gel electrophoresis on RFP.
LAB MANAGERS:Aquire waterproof bandages and vaseline for treatment of minor injuries and to cover minor cuts/rashes/blisters etc.
Aquire hole puncher.
Remember to quiz teammembers regarding MSDS and Lab safety before anyone gets into the lab.
Breef all lab members on general safety procedures in 303 Lab ( the location of MSDS, First Aid, and safety plan).
Gmail correspondence:
Meetings/Notes:
References:
Hairpin loop (Nickel plasmid) possible TF protiens:
SrnQ: http://www.jbc.org/content/278/20/18455.full
Ncrb: http://paperity.org/p/61088047/identification-of-the-transcriptional-regulator-ncrb-in-the-nickel-resistance-determinant
Leaving the lab.jpg
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One of those days....