Template:Kyoto/Notebook/Sep 22

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Sep 22

Colony PCR

Tatsui

Sample	base pair
9/20 Plac-PT181 attenuator(1~4)	664
9/20 Plac-PT181 antisense(1~6)	369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)	859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)	723

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pλ-luxI	225	1.59	0.83

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

Nakamoto

Lane	DNA	Enzyme
1	100bp ladder	--
3	RBS-lacZα-DT	XbaI&PstI
4	RBS-lacZα-DT	XbaI&PstI
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(XbaI&PstI)	9.8	1.50	0.39

digestion

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	100bp ladder	--
3	RBS-lacZα-DT	XbaI&PstI
4	RBS-lacZα-DT	XbaI&PstI
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(XbaI&PstI)	9.8	1.50	0.39

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

Colony PCR

Tatsui

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

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

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/18 Plac (SpeI & PstI)	3.0 µL	9/18 RBS-lysis3-DT (XbaI & PstI)	12.4 µL	3.5 µL
experiment	9/12 Ptet-pT181 antisense	1.0 µL	9/18 spinach-DT	11.9 µL	3.5 µL
experiment	9/13 Pcon-RBS-luxR-DT	2.1 µL	9/10 Plux-RBS-GFP-DT	5.6 µL	3.5 µL
experiment	9/13 Pcon-pT181 attenuator (SpeI & PstI)	2.0 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	6.3 µL	3.5 µL
experiment	9/18 Plac ②(SpeI & PstI)	3.0 µL	9/18 aptamer 12-1R-DT(XbaI & PstI)	4.5µL	x3.5 µL

Liquid Culture

No name

Sample	medium
9/2 spinach-DT-2	--
8/16 Plux-4	--
8/27 Plux-RBS-GFP-DT-1	--
9/5 Pcon-pT181 attenuator-1	--

digestion

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	100bp ladder	--
3	RBS-lacZα-DT	XbaI&PstI
4	RBS-lacZα-DT	XbaI&PstI
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(XbaI&PstI)	9.8	1.50	0.39

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/17 pSB1C3-Pcon-pT181 attenuator-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-antisense-spinach-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-RBS-tetR-DT x2	2 µL	20 µL	CP
9/14 pSB1C3-PkaiBC	2 µL	20 µL	CP
9/14 pSB1C3-RpaB	2 µL	20 µL	CP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-spinach-DT	2 µL	20 µL	CP
9/14 RBS-GFP-DT-PkaiBC	2 µL	20 µL	CP
Plux-RBS-lysis3-DT	2 µL	20 µL	CP
9/20 Ptet-pT181 antisense-spinach-DT	2 µL	20 µL	CP
9/20 Pcon-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 Plac-RBS-lysis3-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer 12_1R	2 µL	20 µL	Amp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT	2 µL	20 µL	Amp
Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20Pcon-pT181attenuator-aptamer-DT	2 µL	20 µL	Amp
9/17 Plac-pT181-attenuatorx2	2 µL	20 µL	Amp
9/15 Pcon-RBS-lacZα-DT	2 µL	20 µL	Amp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT	2 µL	20 µL	Amp
PUC19	2 µL	20 µL	Amp

PCR

Tatsui

Sample	base pair
pT181 attenuator(pSB1C3)	--
Pcon-RBS-lacZα-DT	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 s	30 s	3 min	30 cycles

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/17 pSB1C3-Pcon-pT181 attenuator-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-antisense-spinach-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-RBS-tetR-DT x2	2 µL	20 µL	CP
9/14 pSB1C3-PkaiBC	2 µL	20 µL	CP
9/14 pSB1C3-RpaB	2 µL	20 µL	CP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-spinach-DT	2 µL	20 µL	CP
9/14 RBS-GFP-DT-PkaiBC	2 µL	20 µL	CP
Plux-RBS-lysis3-DT	2 µL	20 µL	CP
9/20 Ptet-pT181 antisense-spinach-DT	2 µL	20 µL	CP
9/20 Pcon-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 Plac-RBS-lysis3-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer 12_1R	2 µL	20 µL	Amp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT	2 µL	20 µL	Amp
Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20Pcon-pT181attenuator-aptamer-DT	2 µL	20 µL	Amp
9/17 Plac-pT181-attenuatorx2	2 µL	20 µL	Amp
9/15 Pcon-RBS-lacZα-DT	2 µL	20 µL	Amp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT	2 µL	20 µL	Amp
PUC19	2 µL	20 µL	Amp

Template:Kyoto/Notebook/Sep 22

From 2013.igem.org

Contents

Sep 22

Colony PCR

Miniprep

Colony PCR

Gel Extraction

digestion

Gel Extraction

Colony PCR

Colony PCR

Colony PCR

Ligation

Liquid Culture

digestion

Gel Extraction

Transformation

PCR

Transformation