Template:Kyoto/Notebook/Sep 9

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Sep 9

PCR

Nakagawa

KaiA(plasmid)	10x buffer	dNTPs	MgSO4	Primer(661-RBS-KaiA-fwd)	Primer(kaiA-662-rev-ver2)	Kod-plus ver2	MilliQ	total
1.0	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

KaiB(plasmid)	10x buffer	dNTPs	MgSO4	Primer(661-RBS-KaiA-fwd-ver2)	Primer(kaiB-663-rev-ver2)	Kod-plus ver2	MilliQ	total
1.0	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

P-KaiBC(PCR products)	10x buffer	dNTPs	MgSO4	Primer(prefix-P-kaiBC-fwd-ver2)	Primer(P-KaiBC-suffix-rev-verw)	Kod-plus ver2	MilliQ	total
0.7	2.5	2.5	1.5	0.75	0.75	0.5	15.8	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

DT(plasmid)	10x buffer	dNTPs	MgSO4	Primer(664-DT-fwd-ver2)	Primer(DT-suffix-660-rev-ver2)	Kod-plus ver2	MilliQ	total
1.0	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

Gel Extraction

Yoshida

Lane	DNA	Enzyme
1	100bp ladder	--
2	KaiA	--
3	KaiA	--
4	KaiB	--
5	KaiB	--
6	P-KaiBC	--
7	P-KaiBC	--
8	DT	--
9	DT	--
10	NC	--
11	100bp ladder	--

Name	concentration[µg/mL]	260/280	260/230
KaiC	28.5	1.26	11.92
Plac	53.9	1.82	1.61
KaiB	59.6	1.61	2.20
KaiB	76.5	1.86	1.44
DT	98.7	1.47	1.43

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/8 pSB1C3	83.2	1.45	0.76
9/8 RBS-lysis2-DT	72.8	1.67	0.97
9/8 spinach-DT	152.6	1.88	1.97
Nuclease-Free water	-0.4	-14.30	0.25

Restriction Enzyme Digestion

No name

	9/9 pSB1C3	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	24µL	1µL	1µL	3µL	3µL	(22)µL	30µL
NC	1.2µL	0µL	0µL	1µL	6.8 µL	10µL

	9/9 pSB1C3	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	27.4µL	1µL	1µL	3µL	3µL	(22)µL	30µL
NC	1.3µL	0µL	0µL	1µL	1µL	6.7µL	10µL

	9/9 spinach-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	10.8µL	1µL	1µL	3µL	3µL	(22)µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
Pcon+RBS-luxI-DT	2µL	20µL	22µL	Amp
Pcon-RBS-luxR-DT+Plux-GFP	2µL	20µL	22µL
Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT	2µL	20µL	22µL

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/8 F1-attenuator(XbaI&PstI)	5.3µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 F3m2-attenuator(XbaI&PstI)	4.5µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 F1-antisense(XbaI&PstI)	1.7µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 F6-antisense(XbaI&PstI)	2.9µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 aptamer12-P(EcoRI&SpeI)	3.1µg	9/8 pSB1C3(EcoRI&SpeI)	8.8µg	3.5µg
experiment	9/8 aptamer12-1M(EcoRI&SpeI)	3.1µg	9/8 pSB1C(EcoRI&SpeI)3	8.8µg	3.5µg

M9 medium

Shimizu & Hirano

EDTA consenlation	Na2HPO4	KH2PO4	NaCl	NH4Cl	Fe(Ⅲ)-EDTA	distilled water	total
1mMol	300mg	150mg	25mg	50mg	21mg	up to 10mL	10mL
10mMol	300mg	150mg	25mg	50mg	210.5mg	up to 10mL	10mL
50mMol	300mg	150mg	25mg	50mg	1052.7mg	up to 10mL	10mL

Colony PCR

No name

Sample	base pair
9/8 Plac-spinach-DT-(1)	659
9/8 Plac-spinach-DT-(2)	659
9/8 Pcon-pt181attenuator-DT-(1)	584

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

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	Spinach-DT	XbaI+PstI
3	Spinach-DT	XbaI+PstI
5	RBS-lacZα-DT	XbaI+PstI
6	RBS-lacZα-DT	XbaI+PstI

failed

Name	quantity	concentration[µg/mL]	260/280	260/230
（´ω｀）
（ΦωΦ^）

Lane	DNA	Enzyme
1	100bp ladder	--
3	pSB1C3	XbaI+PstI
4	pSB1C3	XbaI+PstI

failed

Name	quantity	concentration[µg/mL]	260/280	260/230
（´ω｀）
（ΦωΦ^）

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	pSB1C3(XbaI+PstI)	xx µL	9/8 F1-attenuator(XbaI+PstI)	xx µL	xx µL
experiment	pSB1C3(XbaI+PstI)	xx µL	9/8 F3m2-attenuator(XbaI+PstI)	xx µL	xx µL

PCR

Tatsui and Yoshida

KaiC	10x buffer KOD plus ver2	2M dNTP	MgSO4	Primer Mix(F&R)	Kod-plus ver2	MilliQ	total
0.5	2.5	2.5	1.5	1.5	0.5	16.0	25.0
1.0	2.5	2.5	1.5	1.5	0.5	15.5	25.0
2.0	2.5	2.5	1.5	1.5	0.5	14.5	25.0
4.0	2.5	2.5	1.5	1.5	0.5	12.5	25.0

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

Plac(100pg)	10x buffer KOD plus ver2	2M dNTP	MgSO4	Primer Mix(F&R)	Kod-plus ver2	MilliQ	total
0.3	2.5	2.5	1.5	1.5	0.5	16.2	25.0
0.6	2.5	2.5	1.5	1.5	0.5	15.9	25.0
1.2	2.5	2.5	1.5	1.5	0.5	15.3	25.0
2.4	2.5	2.5	1.5	1.5	0.5	14.1	25.0
0.0	2.5	2.5	1.5	1.5	0.5	16.5	25.0

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

Liquid Culture

No name

Sample	medium
pSB2C3(master)-2	Plusgrow medium(+CP)

Restriction Enzyme Digestion

No name

	8/17 lysis1-2	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	8.1µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/17 lysis3-1	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.1µL	1µL	1µL	3µL	3µL	15.9µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/15 Plac	XbaI	PstI	BSA	Buffer	MilliQ	total
1 cuts	12.1µL	0µL	1µL	3µL	3µL	10.9µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

PCR

Yoshida

KaiA(KaiABC) 100pg/µL	10x buffer KOD plus ver2	dNTPs	MgSO4	Primer Mix(Fwd&Rev)	KOD plus ver2	MilliQ	total
2.0	2.5	2.5	1.5	1.5	0.5	14.5	25
4.0	2.5	2.5	1.5	1.5	0.5	12.5	25
8.0	2.5	2.5	1.5	1.5	0.5	8.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	--
2min	10s	30s	1min	30cycles

PKaiBC 400pg/µL	10x buffer KOD plus ver2	dNTPs	MgSO4	Primer Mix(Fwd&Rev)	KOD plus ver2	MilliQ	total
0.5	2.5	2.5	1.5	1.5	0.5	16.0	25
1.0	2.5	2.5	1.5	1.5	0.5	15.5	25
2.0	2.5	2.5	1.5	1.5	0.5	14.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	--
2m	10s	30s	1m	30cycles

Liquid Culture

No name

Sample	medium
9/2 Spinach-DT	Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT	Plusgrow medium
9/6 master plate	Plusgrow medium(+Amp)
3 Pcon-PT181 attenuator	Plusgrow medium

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	lysis1	XbaI+PstI
4
5
6

Lane	DNA	Enzyme
1	1kbp ladder	--
3	lysis3	XbaI+PstI
4
5
6

Name	concentration[µg/mL]	260/280	260/230
（´ω｀）
（ΦωΦ^）

Electrophoresis

Yoshida

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	Plac 100pg	--	--
3	Plac 200pg	--	--
4	Plac 400pg	--	--
5	Plac 800pg	--	--
6	NC	--	--
7	1kbp ladder	--	--

Template:Kyoto/Notebook/Sep 9

From 2013.igem.org

Contents

Sep 9

PCR

Gel Extraction

Miniprep

Restriction Enzyme Digestion

Transformation

Ligation

M9 medium

Colony PCR

Gel Extraction

Ligation

PCR

Liquid Culture

Restriction Enzyme Digestion

PCR

Liquid Culture

Gel Extraction

Electrophoresis