Template:Kyoto/Notebook/Sep 16

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Sep 16

Liquid Culture

No name

Sample	medium
pSB1C3+P-KaiBC①	Plusgrow medium(+CP)
pSB1C3+P-KaiBC②	Plusgrow medium(+CP)

Colony PCR

No name

Sample	base pair
pSB1C3+P-KaiBC①	283
pSB1C3+P-KaiBC②	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
2min	30s	30s	18s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kb ladder
2	pT181 attenuator	XbaI+PstI
3	pT181 attenuator	XbaI+PstI
4	pT181 attenuator	XbaI+PstI
6	pSB1C3	EcoRI+SpeI
7	pSB1C3	EcoRI+SpeI
8	pSB1C3	EcoRI+SpeI
10	pSB4K5	XbaI+PstI
11	pSB4K5	XbaI+PstI
12	pSB4K5	XbaI+PstI

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator(XbaI+PstI)	8.2	1.62	0.11
pSB1C3(EcoRI+SpeI)	49.5	1.48	0.18
pSB4K5(XbaI+PstI)	18.1	1.73	0.57
DT(SpeI+PstI)	70.4	1.36	1.02
aptamer12-1R-DT(SpeI+PstI)	42.0	1.82	0.47
spinach-DT(SpeI+PstI)	36.8	1.79	0.61

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Dx-lux1	136.7	1.46	1.28
RBS-laczα-DT	237.6	1.88	1.70
pSB4K5	148.4	1.55	1.09
Pcon-KBS-tetR-DT	126.6	1.73	1.36
9/11Plac1	160.0	1.78	1.28
9/15Plac2	144.0	1.73	1.81
9/14Plac	332.5	1.51	2.33
Ptet-RBS-laczα-DT	231.3	1.84	1.57

PCR

No name

DNA	9/6pT181 anti psB1C3(1)	KOD plus	10x buffer	dNTP	MgSO4	GG1_1C3_fwd primer	GG2_1C3_rev primer	MilliQ	total
400pg	1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
800pg	2	0.5	2.5	2.5	1.5	0.75	0.75	14.5	25
1000pg	2.5	0.5	2.5	2.5	1.5	0.75	0.75	14	25
nega	0	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	30s	30cycles

DNA	9/6 12-1R aptamer-DT	KOD plus	10x buffer	dNTP	MgSO4	GG1_1C3_fwd primer	GG1_1C3_rev primer	MilliQ	total
400pg	1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
800pg	2	0.5	2.5	2.5	1.5	0.75	0.75	14.5	25
1000pg	2.5	0.5	2.5	2.5	1.5	0.75	0.75	14	25
NC	0	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	30s	30cycles

Electrophoresis

Yoshida

Lane	Sample
1	100bp ladder
2	antisense 1000pg
3	antisense 800pg
4	antisense 400pg
5	tet aptamer-DT 1000pg
6	tet aptamer-DT 800pg
7	tet aptamer-DT 400pg
8	NC

Restriction Enzyme Digestion

Tatsui

	9/16 Pcon-RBS-tetR-DT(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	15.8	0	0	1	1	3	3	6.2	30
NC	0.8	0	0	0	0	1	1	7.2	10

	9/16 pSB4K5(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	13.5	0	0	1	1	3	3	8.5	30
NC	0.7	0	0	0	0	1	1	7.3	10

	8/22 pSB1C3(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	18.5	1	0	0	1	3	3	3.5	30
NC	0.9	0	0	0	0	1	1	7.1	10

	8/22 pSB1C3(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	18.5	1	1	0	0	3	3	3.5	30
NC	0.9	0	0	0	0	1	1	7.1	10

	8/21 pT181 attenuator2(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	7.4	1	1	0	0	3	3	14.6	30
NC	0.4	0	0	0	0	1	1	7.6	10

Ligation

Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	Pcon-pT181antisense-spinach-DT(XbaI+PstI)	5.0µg/mL	pSB4K5(XbaI+PstI)	18.1µg/mL	xx µg/mL
experiment	pC181 attenuator(XbaI+PstI)	8.2µg/mL	pSB1C3(XbaI+PstI)	26.0µg/mL	xx µg/mL

Transformation

tatsui

Name	Sample	Competent Cells	Total
Pcon+RBS-laczα-DT	µL	µL	µL
Pcon-pT181 attenuator-DT+pSB1C3	µL	µL	µL
pT181 attenuator(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-aptamer12_1R-DT(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-spinach-DT(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-RBS-tetR-DT(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-pT181 antisense-spinach-DT(XbaI&PstI)+pSB1C3	µL	µL	µL

Miniprep

tatsui

DNA	concentration[µg/mL]	260/280	260/230
9/15 pSBiC3	111.7	1.79	1.36
9/15 spinach-DT	113.9	1.57	1.38
9/15 DT	77.0	2.05	1.92

PCR

tatsui

9/14 aptamer12-P(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.5	0.5	1.75	0.5	5.75	10.5

9/14 aptamer12-1M(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.7	0.5	1.75	0.5	6.05	10.5

9/14 Fusion1 attenuator(VF)	Big Dye	5×Buffer	primer	MilliQ	total
2.3	0.5	1.75	0.5	5.45	10.5

9/14 Fusion1 antisense(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.3	0.5	1.75	0.5	6.45	10.5

9/13 Fusion3m2 attenuator(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.7	0.5	1.75	0.5	6.05	10.5

9/13 Fusion6 antisense(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.6	0.5	1.75	0.5	6.15	10.5

9/12 plux(VR)	Big Dye	5×Buffer	primer	MilliQ	total
2.4	0.5	1.75	0.5	5.35	10.5

9/9 Ptet antisense(VF)	Big Dye	5×Buffer	primer	MilliQ	total
2.2	0.5	1.75	0.5	5.55	10.5

9/12 Pcon-pT181 attenuator-DT(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.4	0.5	1.75	0.5	6.35	10.5

9/12 Pcon-pT181 attenuator-DT(VR)	Big Dye	5×Buffer	primer	MilliQ	total
1.4	0.5	1.75	0.5	6.35	10.5

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	60°C	--
2min	10s	5s	4min	30cycles

Template:Kyoto/Notebook/Sep 16

From 2013.igem.org

Contents

Sep 16

Liquid Culture

Colony PCR

Gel Extraction

Miniprep

PCR

Electrophoresis

Restriction Enzyme Digestion

Ligation

Transformation

Miniprep

PCR