Team:Kyoto/Notebook

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template

transformation

じっけんしゃ

Name	Well	Sample	Competent Cells	Total	Plate
いでんし	ぱーつのうぇる	なんちゃらµL	ごにょごにょµL	ほにゃららµL	こーせーぶっしつ
いかどうよう

Aug 19

Colony PCR

Gel Extraction

LB Liquid Mideum Culture

Restriction Enzyme Proccessing

Transformation

Gel Extraction

Master Plate

Miniprep

Aug 20

Miniprep

Kojima

DNA	concentration[µg/µL]	260/280	260/230
8/18 Ptet -1	220	1.69	1.87
8/18 Ptet -2	210	1.69	1.71
8/18 RBS-tetR-DT -1	236	1.63	1.69
8/18 RBS-tetR-DT -2	182	1.65	1.98
8/18 J23100-RBS-GFP-DT -1	334	1.71	2.12
8/18 J23100-RBS-luxR-DT -1	440	1.67	1.60
8/18 J23100-RBS-luxR-DT -2	344	1.68	1.83
8/18 J23100-RBS-lacZα-DT -1	280	1.70	1.81
8/18 RBS-lysis3 -1	282	1.67	1.76
8/18 RBS-lysis3 -2	212	1.30	1.35

LB Culture Medium

Okazaki

volume	200mL
Bacto Trypton	2g
Bacto yeast extract	1g
Nacl	1g
Agar Powder	2g
Amp	40µl

1.Measuring reagents and put they in the Erlenmeyer flask
2.diluting in measuring cylinder to 200ml total
3.autoclaving
4.adding ampicirine 40ml

not written yet

Ristriction Enzyme Proccessing

Kojima and Ashida

8/20 Ptet -1(220µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
4.5	1	1	3	3	17.5	30
0.5	0.2	0	1	1	7.3	10
0.5	0	0.2	1	1	7.3	10
0.5	0	0	1	1	7.5	10

8/20 RBS-tetR-DT -2(182µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
5.5	1	1	3	19.5	30
0.6	0.2	0	1	8.2	10
0.6	0	0.2	1	8.2	10
0.6	0	0	1	8.4	10

8/20 J23100-RBS-GFP-DT(334µg/ml)	EcoRI	SpeI	10x buffer H	MilliQ	total
3.0	1	1	3	22	30
0.3	0.2	0	1	8.5	10
0.3	0	0.2	1	8.5	10
0.3	0	0	1	8.7	10

8/20 J23100-RBS-luxR-DT -2(344µg/mL)	EcoRI	XbaI	BSA	10x buffer M	MilliQ	total
2.9	1	1	3	3	19.1	30
0.3	0.2	0	1	1	7.5	10
0.3	0	0.2	1	1	7.5	10
0.3	0	0	1	1	7.7	10

8/20 RBS-lysis3 -1(282µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
3.5	1	1	3	21.5	30
0.4	0.2	0	1	8.4	10
0.4	0	0.2	1	8.4	10
0.4	0	0	1	8.6	10

8/17 DT -1(188µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
5.3	1	1	3	3	16.7	30
0.3	0.2	0	1	1	7.5	10
0.3	0	0.2	1	1	7.5	10
0.3	0	0	1	1	7.7	10

incubate ~20:00

Transformation

Nakamoto

Name	Sample	Competent Cells(XL10-gold)	Total	Plate
tRNA-Spinach-tRNA	1µL	10µL	11µL	8/19 LB+Amp
tetR-aptamer 12_PC(076)	1µL	10µL	11µL	8/19 LB+Amp
tetR-aptamer 12_1R(113)	1µL	10µL	11µL	8/19 LB+Amp
tetR-aptamer 12_1M(105)	1µL	10µL	11µL	8/19 LB+Amp
pT181 attenuator	1µL	10µL	11µL	8/19 LB+Amp
Fusin1 attenuator	1µL	10µL	11µL	8/19 LB+Amp
Fusion3m2 attenuator	1µL	10µL	11µL	8/19 LB+Amp
pT181 antisense	1µL	10µL	11µL	8/19 LB+Amp
Fusion1 antisense	1µL	10µL	11µL	8/19 LB+Amp
Fusion6 antisense	1µL	10µL	11µL	8/19 LB+Amp

incubate 37°C overnight 20:34~

Electrophoresis

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	8/20 Ptet -1	EcoRI	XbaI
2	8/20 Ptet -1	EcoRI	-
3	8/20 Ptet -1	-	XbaI
4	8/20 Ptet -1	-	-
5	8/20 RBS-tetR-DT -2	EcoRI	SpeI
6	8/20 RBS-tetR-DT -2	EcoRI	-
7	8/20 RBS-tetR-DT -2	-	SpeI
8	8/20 RBS-tetR-DT -2	-	-
9	1kbp ladder	-	-
10	8/20 J23100-RBS-GFP-DT -1	EcoRI	SpeI
11	8/20 J23100-RBS-GFP-DT -1	EcoRI	-
12	8/20 J23100-RBS-GFP-DT -1	-	SpeI
13	8/20 J23100-RBS-GFP-DT -1	-	-
14	8/20 J23100-RBS-luxR-DT -1	EcoRI	XbaI
15	8/20 J23100-RBS-luxR-DT -1	EcoRI	-
16	8/20 J23100-RBS-luxR-DT -1	-	XbaI
17	8/20 J23100-RBS-luxR-DT -1	-	-
18	1kbp ladder	-	-
19	8/20 RBS-lysis3 -1	EcoRI	SpeI
20	8/20 RBS-lysis3 -1	EcoRI	-
21	8/20 RBS-lysis3 -1	-	SpeI
22	8/20 RBS-lysis3 -1	-	-
23	1kbp ladder	-	-
24	8/17 DT	EcoRI	XbaI
25	8/17 DT	EcoRI	-
26	8/17 DT	-	XbaI
27	8/17 DT	-	-

Aug 21

Plusgrow medium(+amp)

Tatsui

material

8/17 Plusgrow medium 50ml
7/22 5000x　Ampicillin 10µl

Measuring materials and putting them in a 50ml tube

Liquid culture

Tatsui

Sample	medium
8/20 tRNA-spinach	8/21 Plusgrow medium(+amp)
8/20 TetR-aptamer 12_P(1076)	8/21 Plusgrow medium(+amp)
8/20 TetR-aptamer 12_1R(113)	8/21 Plusgrow medium(+amp)
8/20 TetR-aptamer 12_1M(105)	8/21 Plusgrow medium(+amp)
8/20 PT181 attenuator	8/21 Plusgrow medium(+amp)
8/20 Fusion1 attenuator	8/21 Plusgrow medium(+amp)
8/20 Fusion3m2 attenuator	8/21 Plusgrow medium(+amp)
8/20 PT181 antisense	8/21 Plusgrow medium(+amp)
8/20 Fusion1 antisense	8/21 Plusgrow medium(+amp)
8/20 Fusion6 antisense	8/21 Plusgrow medium(+amp)

incubate 37°C 10hour

Ristriction Enzyme Proccessing

Kojima and Nakamoto

8/20 Ptet-(1)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
4.5	1	1	3	3	17.5	30
0.5	0.2	0	1	1	7.3	10
0.5	0	0.2	1	1	7.3	10
0.5	0	0	1	1	7.5	10

8/20 Pconst-RBS-luxR-DT-(2)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2.9	1	1	3	3	19.1	30
0.3	0.2	0	1	1	7.5	10
0.3	0	0.2	1	1	7.5	10
0.3	0	0	1	1	7.7	10

8/17 DT	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
5.3	1	1	3	3	16.7	30
0.5	0.2	0	1	1	7.3	10
0.5	0	0.2	1	1	7.3	10
0.5	0	0	1	1	7.5	10

incubate 37°C 1h

Electrophoresis

Aug 22

Liquid culture

Nakamoto

Sample	Medium
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1)	8/21 Plusgrow medium(+amp)
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2)	8/21 Plusgrow medium(+amp)
8/21 Pcon-RBS-GFP-DT control (1)	8/21 Plusgrow medium(+amp)
8/21 Pcon-RBS-GFP-DT control (2)	8/21 Plusgrow medium(+amp)
8/21 RBS-lysis1 (1)	8/21 Plusgrow medium(+amp)
8/21 RBS-lysis1 (2)	8/21 Plusgrow medium(+amp)
8/21 RBS control (1)	8/21 Plusgrow medium(+amp)
8/21 RBS control (2)	8/21 Plusgrow medium(+amp)
8/21 Ptet(pm)-RBS-tetR-DT (1)	8/21 Plusgrow medium(+amp)
8/21 Ptet(pm)-RBS-tetR-DT (2)	8/21 Plusgrow medium(+amp)
8/21 Ptet(pm) control (1)	8/21 Plusgrow medium(+amp)
8/21 Ptet(pm) control	8/21 Plusgrow medium(+amp)
8/21 Plux-RBS-GFP-DT (1)	8/21 Plusgrow medium(+amp)
8/21 pSB1C3(BBa_J04450) (1)	8/21 Plusgrow medium(+amp)
8/21 pSB1C3(BBa_J04450) (2)	8/21 Plusgrow medium(+amp)

incubate 37°C 10hour

Colony PCR

Kojima

Sample	base pair
8/21 RBS-lysis1(1)	400
8/21 RBS-lysis1(2)	400
8/21 RBS control(1)
8/21 RBS control(2)
negative control

94	55	68	cycle
30s	30s	30s	30

Sample	base pair
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1)	2143
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2)	2143
8/21 Pcon-RBS-luxR control(1)
8/21 Pcon-RBS-luxR control(2)
negative control

94	55	68	cycle
30s	30s	30s	30

Sample	base pair
8/21 Ptet(pm)-RBS-tetR-DT (1)
8/21 Ptet(pm)-RBS-tetR-DT (2)
8/21 Ptet(pm) control (1)
8/21 Ptet(pm) control (2)
8/21 Plux-RBS-GFP-DT (1)
8/21 pSB1C3(BBa_J04450) (1)
8/21 pSB1C3(BBa_J04450) (2)
negative control

94	55	68	cycle
30s	30s	30s	30

Team:Kyoto/Notebook

From 2013.igem.org

Contents

template

transformation

Aug 19

Colony PCR

Gel Extraction

LB Liquid Mideum Culture

Restriction Enzyme Proccessing

Transformation

Gel Extraction

Master Plate

Miniprep

Aug 20

Miniprep

LB Culture Medium

Ristriction Enzyme Proccessing

Transformation

Electrophoresis

Aug 21

Plusgrow medium(+amp)

Liquid culture

Ristriction Enzyme Proccessing

Electrophoresis

Aug 22

Liquid culture

Colony PCR