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Wednesday July 6, 2016

Wednesday, 7/6

Members Present: DK, Hamed, Kat, Cathy, Alex, Tam

LAB:

Morning:

- Using Vacufuge to create concentrated RFP plasmids (for training purposes)

- Growing out the cultures further in preparation for mini-prep

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Miniprep using our new NEB monarch kit sample. We miniprepped RFP, and our O2 and O5 overnight cultures.

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Nanodrop of the miniprepped samples.

	A	B	C	D
1	sample	260/280	260/230	concentration (ng/ul)
2	O2?	1.75	0.96	26.7
3	O5?	1.68	0.83	42.1
4	RFP	1.62	0.71	85
5	RFP (vacufuged)	1.73	0.65	69.4

Table1

iGEM (O2)_06_07.TIF

Suspected miniprepped O2 construct.

iGEM O5_06_07.TIF

Suspected miniprepped O5 construct.

iGEM RFP_06_07.TIF

Miniprepped RFP.

iGEM RFP_B1_06_07.TIF

RFP vacufuged and resuspended in 10ul of nuclease free H20.

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Because O2/O5 sample labelling was confused in the first round of miniprep, the samples were miniprepped again:

iGEM O2_2_06_07.TIF

Second attempt at miniprepping the O2 construct.

Afternoon:

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Ran gel of our miniprepped O2, O5, and RFP (these are the properly labelled variants). Also included digested O2 and O5, vacufuged and concentrated RFP, and our previous ligation attempt, L1.

LacZ_O2-5-L1 and RFP (Vac)_July 6, 2016.jpg

GEL: Lane 1: 2-log DNA ladder (NEB) Lane 2: Miniprepped O2. Lane 3: Miniprepped O5. Lane 4: Miniprepped L1 Lane 5: Digested O2 Lane 6: Digested O5 Lane 7: Miniprepped RFP Lane 8: RFP

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Obtained BL21 from Christian -> made 5ml cultures in 2xYPTG and in LB, and also streaked out a LB plate.

For reference:

NEB 2-log ladder.JPG

Administrative:

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Spoke to the NEB guy. Obtained some ladder samples as well as a few miniprep and gel extraction sample kits.

TO DO:

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Digest the O2 with EcoRI and with both EcoRI and PstI to figure out what's up with the weird size of our ligated constructs (should be ~2470bp)

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Run PCR amplification with the rest of our IDT constructs (2 step)

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Run a gel with the questionable O2 and O5.

Team:Toronto/Notebook-w08-wed