Template:Kyoto/Notebook/Sep 5

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Sep 5

Colony PCR

No name

Sample	base pair
8/9 J23100-3	1142
8/9 J23100-4	1142
8/9 J23100-5	1142

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

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	J23100-3	--	--
3	J23100-4	--	--
4	J23100-5	--	--
5	100bp ladder	--	--

File:Igku xx.xxx

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
8/3 J23100-3	349.1	1.69	1.851
8/10 Plac-2	331.3	1.84	1.42

Ligation

Kojima

state	Vector		Inserter		Ligation High ver.2
experiment	Pconst	13.2	spinach-DT	2.1	3.5
experiment	Plac	15.3	spinach-DT	2.1	3.5
experiment	Pconst	13.2	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Plac	15.3	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Ptet	30.1	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Pconst	13.2	pT181 attenuator	11.7	3.5
experiment	Plac	15.3	pT181 attenuator	11.7	3.5
experiment	Ptet	30.1	RBS-lacZα-DT	3.0	3.5

incubate 16 °C 1 hour

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder
2	RBS-luxI-DT
3	RBS-luxI-DT
4	1kbp ladder
5	Pbad/araC
6	Pbad/araC

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (SpeI & PstI)	14.5	1.91	1.09
Plac (SpeI & PstI)	11.6	1.74	1.19
pSB1C3 (EcoRI & SpeI)	5.7	1.74	0.34
pSB1C3 (XbaI & PstI)	5.6	2.00	0.37

Colony PCR

No name

Sample	base pair
9/4 apt12_1R(pSB1C3)-1	384
9/4 apt12_1R(pSB1C3)-2	384
9/4 pT181 antisense(pSB1C3)-1	415
9/4 pT181 antisense(pSB1C3)-2	415
9/4 pT181 antisense(pSB1C3)-3	415
9/4 pT181 antisense(pSB1C3)-4	415

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

Sample	base pair
9/4 apt12_1R-DT-1	521
9/4 apt12_1R-DT-2	521
9/4 apt12_1R-DT-3	521
9/4 apt12_1R-DT-4	521
9/4 pT181 attenuator(pSB1C3)	601
9/4 pT181 attenuator(pSB1C3)	601
9/4 RBS-lysis1-DT	613
9/4 RBS-lysis1-DT	613

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	36s	30cycles

Sample	base pair
9/4 RBS-lysis2-DT-1	985
9/4 RBS-lysis2-DT-2	985
9/4 RBS-lysis2-DT-3	985
9/4 RBS-lysis2-DT-4	985
9/4 RBS-lysis3-DT-1	1210
9/4 RBS-lysis3-DT-2	1210

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	72s	30cycles

Liquid Culture

Nakamoto

Sample	medium
8/9 J23100-5	Plusgrow medium(+Amp)

37°C

Transformation

じっけんしゃ

Name	Sample(µL)	Competent Cells(µL)	Total(µL)	Plate
9/5 Pconst+spinach-DT	2	20	22	--
9/5 Plac+spinach-DT	2	20	22	--
9/5 Pconst+pT181 antisense	2	20	22	--
9/5 Plac+pT181 antisense	2	20	22	--
9/5 Ptet+pT181 antisense	2	20	22	--
9/5 Plux+pT181 attenuater	2	20	22	--
9/5 Ptet+pT181 attenuater	2	20	22	--
9/5 Ptet+pT181 attenuater	2	20	22	--
9/5 Pconst+RBS-tetR+DT	2	20	22	--
9/16(2012) T7-His-FT	2	20	22	--
9/16(2012) pBr322	2	20	22	--

Restriction Enzyme Digestion

じっけんしゃ

	9/8 Pcon-pT181 anti	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	8.2µL	0µL	1µL	0µL	1µL	3µL	3µL	13.8µL	30µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/8 Pcon-spin-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	7.9µL	1µL	1µL	0µL	0µL	3µL	3µL	14.1µL	30µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/21 F1 atte	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.5µL	0µL	0µL	1µL	1µL	3µL	3µL	15.5µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F3m2 atte	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	0µL	1µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F1 anti	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.7µL	0µL	0µL	1µL	1µL	3µL	3µL	15.3µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F6 anti	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	0µL	1µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 tetR aptamer 12_1M	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	5.3µL	1µL	1µL	0µL	0µL	3µL	3µL	16.7µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 tetR aptamer 12_P	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	13.3µL	1µL	1µL	0µL	0µL	3µL	3µL	8.7µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/4 spin-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	5.4µL	0µL	0µL	1µL	1µL	3µL	3µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/7 RBS-lysis1-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	10µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/6 RBS-lysis2-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	13µL	0µL	0µL	1µL	1µL	3µL	3µL	9µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/7 RBS-lysis3-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	10µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 J23100 RBS-lacZα-DT-(1)	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	7µL	0.5µL	0.5µL	0µL	0µL	3µL	3µL	16µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 RBS-lacZα-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	10µL	0.5µL	0.5µL	0µL	0µL	3µL	3µL	13µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

5*M9(EDTA)

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis1-DT	XbaI & PstI
3	RBS-lysis1-DT	XbaI & PstI
5	RBS-lysis3-DT	XbaI & PstI
6	RBS-lysis3-DT	XbaI & PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis1-DT (XbaI & PstI)	5.3	1.98	0.06
RBS-lysis3-DT (XbaI & PstI)	5.3	1.98	0.06

Lane	DNA	Enzyme
1	100bp ladder
3	Pcon-pT181 attenuator	EcoRI & SpeI
4	Pcon-pT181 attenuator	EcoRI & SpeI
6	Spinach-DT	XbaI & PstI
7	Spinach-DT	XbaI & PstI
9	RBS-lacZα-DT	XbaI & PstI
10	RBS-lacZα-DT	XbaI & PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (EcoRI & SpeI)	5.2	1.82	0.36
Spinach-DT (XbaI & PstI)	8.0	1.87	0.25
RBS-lacZα-DT (XbaI & PstI)	10.3	1.71	0.54

Lane	DNA	Enzyme
1	1kbp ladder
2	Ptet-pT181 antisense	EcoRI & PstI
3	Ptet-pT181 antisense	EcoRI & PstI
5	pSB4K5	EcoRI & SpeI
6	pSB4K5	EcoRI & SpeI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Name	concentration[µg/mL]	260/280	260/230
Ptet-pT181 antisense(EcoRI & PstI)	30.6	1.84	1.16
pSB4K5 (EcoRI & SpeI)	21.8	1.87	0.98

Lane	DNA	Enzyme
1	1kbp ladder
2	Pcon-pT181 attenuator	SpeI & PstI
3	Pcon-pT181 attenuator	SpeI & PstI
5	Plac	SpeI & PstI
6	Plac	SpeI & PstI
8	pSB1C3	EcoRI & SpeI
9	pSB1C3	EcoRI & SpeI
11	pSB1C3	XbaI & PstI
12	pSB1C3	XbaI & PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (SpeI & PstI)	14.5	1.91	1.09
Plac (SpeI & PstI)	11.6	1.74	1.19
pSB1C3 (EcoRI & SpeI)	5.7	1.74	0.34
pSB1C3 (XbaI & PstI)	5.6	2.00	0.37

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pcon-pT181 anti	243.0	1.90	1.81
Pcon-Spinach-DT	253.7	1.57	1.51

Colony PCR

No name

Sample	base pair
9/8 Plac-spinach-DT-(1)	659

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5m	30s	30s	40s	30cycles

Sample	base pair
9/8 Plac-spinach-DT-(2)	659

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5m	30s	30s	40s	30cycles

Sample	base pair
9/8 Pcon-pt181atte-DT-(1)	584

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5m	30s	30s	40s	30cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/19 lysis1	XbaI	PstI
3	8/19 lysis1	XbaI	--
4	8/19 lysis1	--	PstI
5	8/19 lysis1	--	--
6	8/19 lysis1(Gel Extraction Product)	XbaI	PstI

Liquid Culture

Hirano

Sample	medium
JM109-1(Master plate)	LB medium

Template:Kyoto/Notebook/Sep 5

From 2013.igem.org

Contents

Sep 5

Colony PCR

Electrophoresis

Miniprep

Ligation

Gel Extraction

Colony PCR

Liquid Culture

Transformation

Restriction Enzyme Digestion

5*M9(EDTA)

Gel Extraction

Miniprep

Colony PCR

Electrophoresis

Liquid Culture