Template:Kyoto/Notebook/Sep 17

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Revision as of 05:56, 26 September 2013

Sep 17

Liquid Culture

Tatsui and Hirano

Sample	medium
Spinach-DT	LB medium(+CP)
Pcon-Spinach-DT	LB medium(+Amp)
Pcon-pT181 antisense-Spinach-DT	LB medium(+Amp)

PCR

Hirano

Pcon-tetR aptamer-DT(VF)	BigDye	5x buffer	primer(VF)	MilliQ	total
1.3	0.5	1.75	0.5	0.45	10.5

Pcon-tetR aptamer-DT(VR)	BigDye	5x buffer	primer(VR)	MilliQ	total
1.3	0.5	1.75	0.5	0.45	10.5

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

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pcon-RBS-tetR-DT	XbaI	PstI
3	Pcon-RBS-tetR-DT	--	--
4	pSB4K5	XbaI	PstI
5	pSB4K5	--	--
6	pSB1C3	EcoRI	SpeI
7	pSB1C3	EcoRI	PstI
8	pSB1C3	--	--
9	pT181 attenuator	EcoRI	SpeI
10	pT181 attenuator	--	--
11	1kbp ladder	--	--

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	8/28 Plux (SpeI & PstI)	5.0 µL	9/13 RBS-lysis1-DT (XbaI & PstI)	3.5 µL	3.5 µL
experiment	8/28 Plux (SpeI & PstI)	5.0 µL	9/13 RBS-lysis2-DT (XbaI & PstI)	3.6 µL	3.5 µL
experiment	8/28 Plux (SpeI & PstI)	11.0 µL	9/13 RBS-lysis3-DT (XbaI & PstI)	9.5 µL	3.5 µL
experiment	9/14 Plac (SpeI & PstI)	2.4 µL	9/16 pT181 attenuator-DT (XbaI & PstI)	4.8 µL	3.5 µL
experiment	9/14 Plac (SpeI & PstI)	2.4 µL	9/3 pT181 antisense (XbaI & PstI)	2.0 µL	2.2 µL
experiment	9/14 Plac (SpeI & PstI)	2.4 µL	9/10 aptamer 12_1R-DT (XbaI & PstI)	1.8 µL	2.1 µL
experiment	9/13 pSB1C3 (XbaI & PstI)	2.0 µL	9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI)	12.0 µL	3.5 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-Spinach-DT (EcoRI & SpeI)	4.1 µL	2.5 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)	2.0 µL	1.5 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI)	2.4 µL	1.7 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI)	2.0 µL	1.5 µL
experiment	9/13 pSB1C3 (XbaI & PstI)	2.0 µL	9/16 Pcon-pT181-attenuator (XbaI & PstI)	4.4 µL	3.2 µL

Gel Extraction

No Name

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pT181 attenuator -(2)	EcoRI & SpeI
3
4

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

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator (EcoI & SpeI)	9.2	1.15	0.12

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI & SpeI
3
4

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

Name	concentration[µg/mL]	260/280	260/230
pSB1C3	10.0	1.84	0.40

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB4K5	XbaI & PstI
3
4

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

Name	concentration[µg/mL]	260/280	260/230
pSB4K5	7.6	1.81	0.32

Lane	DNA	Enzyme
1	1kbp ladder	--
2	Pcon-RBS-tetR-DT	XbaI & PstI
3
4

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

Name	concentration[µg/mL]	260/280	260/230
Pcon-RBS-tetR-DT	8.0	2.40	1.14

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI & PstI
3
4

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

Name	concentration[µg/mL]	260/280	260/230
pSB1C3	9.1	2.17	0.35

Mutation PCR

Kinose

pC KaiABC(100pg/µL)	Prime STAR(2x)	Primer(KaiA-BsaI MuT-Fwd)	Primer(KaiA-BsaI MuT-Rev)	MilliQ	total
1.0	25.0	1.0	1.0	22.0	50.0
1.5	25.0	1.0	1.0	21.5	50.0
2.0	25.0	1.0	1.0	21.0	50.0

PreDenature	Denature	Annealing	Extension	cycle
96 °C	98 °C	65 °C	72 °C	--
5 min	10 s	15 s	40 s	30 cycles

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Template:Kyoto/Notebook/Sep 17

From 2013.igem.org

Revision as of 05:56, 26 September 2013

Contents

Sep 17

Liquid Culture

PCR

Electrophoresis

Ligation

Gel Extraction

Mutation PCR