Talk:20.109(F09): Mod 2 Day 6 Readouts of DNA, Protein

Information about genetic screen

T/R lab

INFORMATION TO BE FILLED IN BY EACH GROUP

team color	library screened (K+ or P+)	Approx # of colonies examined (i.e., the # of colonies you looked at to find your 2 candidates)	Seq data	b-gal data	Notes
Red	P+		Mutant 2 carries two mutations: A553I and H557A	Mutant 1 -- Mutant 2 -- 366+HA -- 366-HA Light 65 -- 66 -- 201 -- 232 Dark 53 -- 25 -- 170 -- 1340	* Borrowed Mutant 2 from Green candidate 2
Orange	K+	2	Mutant 1 did not generate sequence data. Mutant 2 carries two mutations: G554V, V555S, S556A	WT without HA Light Average= 567 Dark Average= 961 WT-HA Tagged Light 3 replicates: 221.9531881 -- 212.4645892 -- 233.3603896 Average= 222.592722 STDEV= 10.46257002 Dark 3 replicates: 1588.465298 -- 174.3015663 -- 168.576792 Average= 643.7812188 STDEV= 818.1254186 Mutant 2 Light 3 replicates: 521.467 -- 526.889 -- 507.7317 STDEV= 9.87505632370193 Average= 518.696264320771 Dark 3 replicates: 684.342 -- 668.203 -- 684.615 STDEV= 9.39776470423833 Average= 679.053216786013
Yellow	P+	2
Green
Blue
Pink	K+	2
Purple
Silver

All mutant candidates were digested with a cocktail of Nde and Mlu in 1X NEB3 for 45 minutes at 37° then loaded on a 1% agarose gel (1XTAE)

T/R Red, Orange, Yellow, Green

Lane	Team	Sample
1		1Kb DNA Ladder
2	Red	candidate 1 Nde/Mlu digest
3		candidate 2 Nde/Mlu digest
4		blank
5		1Kb DNA Ladder
6	Orange	candidate 1 Nde/Mlu digest
7		candidate 2 Nde/Mlu digest
8		blank
9		1Kb DNA Ladder
10	Yellow	candidate 1 Nde/Mlu digest
11		candidate 2 Nde/Mlu digest
12		1Kb DNA Ladder
13	Green	candidate 1 Nde/Mlu digest
14		candidate 2 Nde/Mlu digest

T/R Blue, Pink, Purple, Control digests

Lane	Team	Sample
1		1Kb DNA Ladder
2	Blue	candidate 1 Nde/Mlu digest
3		candidate 2 Nde/Mlu digest
4		1Kb DNA Ladder
5	Pink	candidate 1 Nde/Mlu digest
6		candidate 2 Nde/Mlu digest
7		1Kb DNA Ladder
8	Purple	candidate 1 Nde/Mlu digest
9		candidate 2 Nde/Mlu digest
10		blank
11		1Kb DNA Ladder
12	Control with stuffer fragment	Nde/Mlu digest
13	Control without stuffer fragment	Nde/Mlu digest

Here is your Western data. If you'd like to see the blots themselves, they'll be in the lab at the front bench.
The "?" after your team color are associated with teams who labeled their containers on the lids rather than the tubs. These blots were possibly mis-designated during the washing and scanning of these blots since the lids were separated from the tubs.
If you'd like to note which of the 2 preparations of primary antibody you used, you can do so in the label under the blot's image

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  Red Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

• 

  Orange Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

• 

  Yellow? Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

• 

  Green? Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

• 

  Blue? Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

• 

  Pink Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

• 

  Purple? Western, probed with mouse raised monoclonal anti-HA, detection with Goat antimouse + AP

W/F lab

Digest Sample info

Please fill in the info below for your team for others to refer to:

team color	library screened (K+ or P+)	Approx # of colonies examined	Seq data	b-gal data	Notes
Red
Orange
Yellow
Green
Blue	P+	2 red group's Colonies		Mutant 1 Mutant 2 366 Light/n 17.4 47.5 163.1 18.3 54.4 157.1 Average 17.85 50.95 160.1 Stdev 0.636396103 4.87903679 4.242640687 Dark 18.9 43.6 178.5 18.6 44.7 190.6 Average 18.75 44.15 184.55 Stdev 0.212132034 0.777817459 8.555992052
Pink	K+	2	:(	Mutant 1 Light (Avg): 144 Mutant 1 Dark (Avg): 143 Mutant 2 Light (Avg): 327 Mutant 2 Dark (Avg): 301
Purple	Using Red Group's Data (sequencing failed)	N/A
Silver

Digest Images

Candidates were digested for 55' at 37 °C and run on a 1% agarose gel. Each gel was photographed at two different exposure times - use whichever one makes your DNA easier to see. Also see the second T/R gel for how control DNA (with and without stuffer fragment) runs.

Gel 1 Lane	Gel 1 Sample	Gel 1 Lane	Gel 1Sample
1	1 Kb marker	6	Green Cand 1
2	Red Cand 1	7	Green Cand 2
3	Red Cand 2	8
4		9
5	1 Kb marker	10

Gel 2 Lane	Gel 2 Sample	Gel 2 Lane	Gel 2Sample
1	1 Kb marker	6	Pink Cand 1
2	Blue Cand 1	7	1 Kb marker
3	Blue Cand 2	8	Purple Cand 1
4	1 Kb marker	9	Purple Cand 2
5	Pink Cand 1	10

Blue, Pink, Purple lower exposure

Blue, Pink, Purple higher exposure

Red, Green lower exposure

Red, Green higher exposure

Western images

Below are your Westerns. (Please crop out your image for use in the research article.) Some information may be lost in the digital images, so feel free to look at the original blots after journal club to get a good sense of where your bands are.

Westerns