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

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

2X - 20pg.jpg

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

- Made 1ml aliquots of nuclease free water, stored on a rack on the shelf in 403

- Miniprepped RFP:

RFP_1.jpg

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

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 - MP1A	1.63	1.27	16.7 ng/uL
3	RFP - MP1B	1.43	0.61	15.0 ng/uL	used for transformation
4	RFP - MP2A	1.59	0.6	18.2 ng/uL
5	RFP - MP2B	1.55	0.65	12.8 ng/uL

Table1

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Eluted with nuclease free water, and blanked the nanodrop woth nuclease free water

Afternoon:

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Obtained chemically competent cells from Kayla:

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Followed the bacterial transformation protocol, set up the following transformations:

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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		CCA	CCB	CC0
3	DNA added:	0.5pg	0.5pg	50pg
4		10pg	10pg	RFP1B
5		50pg	50pg
6		RFP1B	RFP1B
7		Control - no plasmid	Control - 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)

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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:

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SrnQ: http://www.jbc.org/content/278/20/18455.full

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Ncrb: http://paperity.org/p/61088047/identification-of-the-transcriptional-regulator-ncrb-in-the-nickel-resistance-determinant

Leaving the lab.jpg

One of those days....

Team:Toronto/Notebook-w05-thu