Template:Kyoto/Notebook/Aug 22

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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 (2)	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	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

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	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycles

Sample	base pair
8/21 Ptet-RBS-tetR-DT (1)	1216
8/21 Ptet-RBS-tetR-DT (2)	1216
8/21 Ptet control (1)	--
8/21 Ptet control (2)	--
8/21 Plux-RBS-GFP-DT (1)	1227
8/21 pSB1C3(BBa_J04450) (1)	1353
8/21 pSB1C3(BBa_J04450) (2)	1353
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	pT181antisense	EcoRI	SpeI
2	pT181antisense	EcoRI	--
3	pT181antisense	--	SpeI
4	pT181antisense	--	--
5	pT181antisense	XbaI	PstI
6	pT181antisense	XbaI	--
7	pT181antisense	--	PstI
8	pT181antisense	--	--
9	100bp ladder	--	--
10	Fusion1 attenuator	EcoRI	SpeI
11	Fusion1 attenuator	EcoRI	--
12	Fusion1 attenuator	--	SpeI
13	Fusion1 attenuator	--	--
14	Fusion1 attenuator	XbaI	PstI
15	Fusion1 attenuator	XbaI	--
16	fusion1 attenuator	--	PstI
17	fusion1 attenuator	--	--

File:Igku xxxxxx.xxx

Lane	Sample	Enzyme1	Enzyme2
1	Fusion3 3m2 attenuator	EcoRI	SpeI
2	Fusion3 3m2 attenuator	EcoRI	--
3	Fusion3 3m2 attenuator	--	SpeI
4	Fusion3 3m2 attenuator	--	--
5	Fusion3 3m2 attenuator	XbaI	PstI
6	Fusion3 3m2 attenuator	XbaI	--
7	Fusion3 3m2 attenuator	--	PstI
8	Fusion3 3m2 attenuator	--	--
9	100bp ladder	--	--
10	aptamer 12-1M	EcoRI	SpeI
11	aptamer 12-1M	EcoRI	--
12	aptamer 12-1M	--	SpeI
13	aptamer 12-1M	--	--
14	aptamer 12-P	EcoRI	SpeI
15	aptamer 12-P	EcoRI	--
16	aptamer 12-1P	--	SpeI
17	aptamer 12-1P	--	--

File:Igku xxxxxx.xxx

Restriction Enzyme Digestion

Tatsui, Kojima, Honda and Ashida

Fusion1 antisense (1) (300 µg/mL)	EcoRI	SpeI	buffer H	MilliQ	total
3.3	1	1	3	21.7	30
0.7	0.2	0	1	8.1	10
0.7	0	0.2	1	8.1	10
0.7	0	0	1	8.3	10

Fusion1 antisense (1) (300 µg/mL)	XbaI	PstI	buffer M	BSA	MilliQ	total
3.3	1	1	3	3	18.7	30
0.7	0.2	0	1	1	7.1	10
0.7	0	0.2	1	1	7.1	10
0.7	0	0	1	1	7.3	10

Fusion6 antisense (1) (336 µg/mL)	EcoRI	SpeI	buffer H	MilliQ	total
3	1	1	3	22	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

Fusion6 antisense (1) (336 µg/mL)	XbaI	PstI	buffer M	BSA	MilliQ	total
3	1	1	3	3	19	30
0.6	0.2	0	1	1	7.2	10
0.6	0	0.2	1	1	7.2	10
0.6	0	0	1	1	7.4	10

pT181 antisense (1) (178 µg/mL)	EcoRI	SpeI	buffer H	MilliQ	total
5.6	1	1	3	19.4	30
1.1	0.2	0	1	7.7	10
1.1	0	0.2	1	7.7	10
1.1	0	0	1	7.9	10

pT181 antisense (1) (178 µg/mL)	XbaI	PstI	buffer M	BSA	MilliQ	total
5.6	1	1	3	3	16.4	30
1.1	0.2	0	1	1	6.7	10
1.1	0	0.2	1	1	6.7	10
1.1	0	0	1	1	6.9	10

pT181 attenuator (1)	EcoRI	SpeI	buffer H	MilliQ	total
3	1	1	3	22	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

pT181 attenuator (1)	XbaI	PstI	buffer M	BSA	MilliQ	total
3	1	1	3	3	19	30
0.6	0.2	0	1	1	7.2	10
0.6	0	0.2	1	1	7.2	10
0.6	0	0	1	1	7.4	10

Fusion1 attenuator (1)	EcoRI	SpeI	buffer H	MilliQ	total
3.3	1	1	3	21.7	30
0.7	0.2	0	1	8.1	10
0.7	0	0.2	1	8.1	10
0.7	0	0	1	8.3	10

Fusion1 attenuator (1)	XbaI	PstI	buffer M	BSA	MilliQ	total
3.3	1	1	3	3	18.7	30
0.7	0.2	0	1	1	7.1	10
0.7	0	0.2	1	1	7.1	10
0.7	0	0	1	1	7.3	10

Fusion3m2 attenuator (1)	EcoRI	SpeI	buffer H	MilliQ	total
3	1	1	3	22	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

Fusion3m2 attenuator (1)	XbaI	PstI	buffer M	BSA	MilliQ	total
3	1	1	3	3	19	30
0.6	0.2	0	1	1	7.2	10
0.6	0	0.2	1	1	7.2	10
0.6	0	0	1	1	7.4	10

Spinach (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.3	0.5	0.5	3	0.3	23.4	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_P (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_1R (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.6	0.5	0.5	3	0.3	23.1	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_1M (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

J23100	SpeI	PstI	buffer B	BSA	MilliQ	total
7	0.5	0.5	3	0.3	18.7	30
1.4	0.1	0	1	0.1	7.4	10
1.4	0	0.1	1	0.1	7.4	10
1.4	0	0	1	0.1	7.5	10

Plac (2)	SpeI	PstI	buffer B	BSA	MilliQ	total
11.2	0.5	0.5	3	0.3	14.5	30
2.2	0.1	0	1	0.1	6.6	10
2.2	0	0.1	1	0.1	6.6	10
2.2	0	0	1	0.1	6.7	10

Miniprep

Ashida

DNA	concentration[µg/mL]	260/280	260/230
8/22 RBS-lysis (1)	128	1.77	2.29
8/22 RBS-lysis (2)	96	1.80	2.07
8/22 pSB1C3 (1)	108	1.55	1.45
8/22 pSB1C3 (2)	130	1.69	1.76

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	8/22 pT181 attenuator (1)	EcoRI	SpeI
3	8/22 pT181 attenuator (1)	EcoRI	--
4	8/22 pT181 attenuator (1)	--	SpeI
5	8/22 pT181 attenuator (1)	--	--
6	8/22 pT181 attenuator (1)	XbaI	PstI
7	8/22 pT181 attenuator (1)	XbaI	--
8	8/22 pT181 attenuator (1)	--	PstI
9	8/22 pT181 attenuator (1)	--	--
10	8/22 Fusion6 antisense (1)	EcoRI	SpeI
11	8/22 Fusion6 antisense (1)	EcoRI	--
12	8/22 Fusion6 antisense (1)	--	SpeI
13	8/22 Fusion6 antisense (1)	--	--
14	100bp	--	--

File:Igku xxxxxx.xxx

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	8/22 Fusion6 antisense (1)	XbaI	PstI
3	8/22 Fusion6 antisense (1)	XbaI	--
4	8/22 Fusion6 antisense (1)	--	PstI
5	8/22 Fusion6 antisense (1)	--	--
6	8/22 Fusion1 antisense (1)	EcoRI	SpeI
7	8/22 Fusion1 antisense (1)	EcoRI	--
8	8/22 Fusion1 antisense (1)	--	SpeI
9	8/22 Fusion1 antisense (1)	--	--
10	8/22 Fusion1 antisense (1)	XbaI	PstI
11	8/22 Fusion1 antisense (1)	XbaI	--
12	8/22 Fusion1 antisense (1)	--	PstI
13	8/22 Fusion1 antisense (1)	--	--
14	100bp	--	--

File:Igku xxxxxx.xxx

tetR aptamer 12_P (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_1M (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_P (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_1M (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

Template:Kyoto/Notebook/Aug 22

From 2013.igem.org

Contents

Aug 22

Liquid culture

Colony PCR

Electrophoresis

Restriction Enzyme Digestion

Miniprep

Electrophoresis

tetR aptamer 12_P (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10

tetR aptamer 12_1M (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2.7	0.5	0.5	3	0.3	23	30
0.5	0.1	0	1	0.1	8.3	10
0.5	0	0.1	1	0.1	8.3	10
0.5	0	0	1	0.1	8.4	10