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Tuesday July 12th

Tuesday, 7/12

Members Present: Hamed, Celine, Kat, Bohdan, Marc, Karim

LAB:

Morning:

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Miniprep of transformed & cultured "ligated" samples from the previous day of: (recall some colonies appeared red - we suspect that this is RFP contamination) We will run a gel on these tomorrow to verify band size. {see July11th entry for pictures of the plates that we are mentioning}

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GolS (red and white colonies)

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GolS P118A (red colony)

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mCherry (white colony)

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Subsequent nanodrop of the aforementioned minipreped samples:

	A	B	C	D
1	Sample	260/280	260/230	concentration (ng/ul)
2	GolS - from red colony	2.12	1.04	16.7
3	GolS - from white colony	1.86	1.1	20
4	GolS P118A	1.67	1.03	21.3
5	mCherry	1.77	1.25	22.7

Table1

GolS-Red.TIF

Nanodrop of our "ligated" and miniprepped GolS. Note that the colony obtained was red, and therefore we suspect that it is RFP.

GolS-White.TIF

Nanodrop of our "ligated" and miniprepped GolS. Note that the colony obtained was white amidst red colonies.

mCherry.TIF

Nanodrop of our "ligated" and miniprepped mCherry. Note that the colony obtained was not red (while it should have been) therefore we suspect that this construct was not successfully ligated.

P118A.TIF

Nanodrop of our "ligated" and miniprepped GolS P118A. Note that the colonies on this plate were all red, making us believe that it is in fact RFP contamination.

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Obtained new T4 ligase and ligase buffer from medstore

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Hamed created a Ligation Calculator for our restriction enzyme digest and ligation, which follows NEB's guidelines. Previously we were using iGEM's protocols.

Ligation-Calculator-iGEM.xlsx

Afternoon:

We decided to do a mass restriction enzyme digestion and ligation of all of our constructs (we have new T4 ligase + buffer):

1.

TetO-LacZ

2.

TetO-mCherry

3.

pgolB-LacZ

4.

Long GolSP118A

5.

Short Teto GolS P118A

6.

Long GolS

7.

Short TetO GolS

8.

Short PpcnA LacZ

9.

Short TetO RcnR

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We then directly transformed with the obtained ligated parts. Note that the numbering code above also corresponds to the numbering code that can be found on the plated transformants (ie L1 refers to ligated TetO-LacZ)

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Considering that our transformation protocol yields about ~200ul of product, we made replicates of each ligation and labelled them as A and B (going with the above example - both L1A and L1B plates can be found)

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We also transformed RFP as a positive control, as per usual.

TO DO:

For the next day:

LAB TEAM:

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Run a gel on the miniprepped and nanodropped samples:

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GolS (red)

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GolS (white)

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GolS P118A (red)

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mCherry (white colony)

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RFP (any one of our previously miniprepped samples should do)

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2-log ladder (no more than 6ul)

Keep in mind that we suspect that the red colonies are RFP contaminants and should have the same size as the RFP plasmid (see our parts catalogue for construct sizes). mCherry actually is pretty similar in size (and coincidentally also red and fluorescent) so we can't discern the two.

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Make 5ml overnight cultures of any transformants that should appear on any of the L1-L9 plates. Keep in mind that we transformed fairly late in the evening so you might want to wait until the end of the day to select for colonies. Keep in mind to sign the logbook for the shaker, too. If there are no colonies proceed to WB242 and cry.

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Grow the BL21 cells to OD600 ~3.0. They were put in Shaker 2 at 300rpm (which is kinda freaky to watch) according to Christian's recommendations. Take OD measurements throughout the day, using the spec in 319. There is some 2xYTPG left on the autoclave shelf for spec blanks, and pre-cut parafilm squares can be found in the upper left breast pocket of Kat's lab coat.

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Make more LB+ CAM and LB plates because we never have enough, apparently.

LAB MANAGERS:

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Figure out how to dispose of the sharps in 403 (possibly look into procuring a different sharps container - perhaps talk to Susie about that)

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Purchases made today:

	A	B	C	D	E	F
1	Name	SKU	Unit Price	Quantity	Total (incl. tax)	Status
2	T4 DNA Ligase - 20,000 units	M0202S	80.64	1	91.12	available
3	Large Cylinder and Bottle Brush	70230-018	3.56	1	4.02	available