Template:Kyoto/Notebook/Sep 25

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Sep 25

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12)	859
9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8)	1527
9/23 Pcon-PT181 attenuator-pSB1C3-(5~8)	601

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
2 min	30 s	30 s	1min5s	30 cycles

Electrophoresis

no name

Lane	Sample	Enzyme1	Enzyme2
1	Pcon-PT181 attenuator-aptamer12-1R-DT-5	--	--
2	Pcon-PT181 attenuator-aptamer12-1R-DT-6	--	--
3	Pcon-PT181 attenuator-aptamer12-1R-DT-7	--	--
4	Pcon-PT181 attenuator-aptamer12-1R-DT-8	--	--
5	Pcon-PT181 attenuator-aptamer12-1R-DT-9	--	--
6	Pcon-PT181 attenuator-aptamer12-1R-DT-10	--	--
7	Pcon-PT181 attenuator-aptamer12-1R-DT-11	--	--
8	Pcon-PT181 attenuator-aptamer12-1R-DT-12	--	--
9	1kbp ladder	--	--
10	Pcon-PT181 attenuator-RBS-GFP-DT-5	--	--
11	Pcon-PT181 attenuator-RBS-GFP-DT-6	--	--
12	Pcon-PT181 attenuator-RBS-GFP-DT-7	--	--
13	Pcon-PT181 attenuator-RBS-GFP-DT-8	--	--
14	Pcon-PT181 attenuator-pSB1C3-5	--	--
15	Pcon-PT181 attenuator-pSB1C3-6	--	--
16	Pcon-PT181 attenuator-pSB1C3-7	--	--
17	Pcon-PT181 attenuator-pSB1C3-8	--	--

Transformation

kojima and nakamoto

Name	Sample	Competent Cells	Total	Plate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	-
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	-

Name	Sample	Competent Cells	Total	Plate
aptamer12-DT(EcoRI+Xbal)+Pcon-pT181attenuator(EcoRI+Spel)	2µL	20µL	22µL	-
Ptet(Spel+Pstl)+RBS-GFP-DT(Xbal+Pstl))	2µL	20µL	22µL	-

Ligation

nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/25 aptamer12_1R-DT(EcoRI&XbaI)	4.5 µL	Pcon-pT181 attenuator(EcoRI&SpeI)	7.7 µL	6.1 µL
experiment	9/17 pSB1C3(EcoRI&SpeI)	2.0 µL	9/3 pT181 attenuator(EcoRI&SpeI)	14 µL	8 µL
experiment	9/25 Pcon-pT181 attenuator(EcoRI&XbaI)	4.5 µL	9/25 RBS-GFP-DT(EcoRI&SpeI)	19.3 µL	11.9 µL
experiment	9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)	4.2 µL	Pcon-RBS-tetR-DT(EcoRI&SpeI)	20 µL	12.1 µL

Digestion

Liquid Culture

Kojima

Sample	medium
DT	-
pT181attenuator	-
Pcon-RBS-tetR-DT	-

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	100bp ladder	-
2	Pcon-pT181antisense
3
4
5	-
6	DT
7
8
9	-
10	Spinach-DT
11
12

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

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

Lane	DNA	Enzyme
1
2
3
4
5
6

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

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

Lane	DNA	Enzyme
1
2
3
4
5
6

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

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

Lane	DNA	Enzyme
1
2
3
4
5
6

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

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

Lane	DNA	Enzyme
1
2
3
4
5
6

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

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

Nakamoto

Lane	DNA	Enzyme
1
2
3
4
5
6

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

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

Restriction Enzyme Digestion

Tatsui

	DNA	restricion enzyme	restriction enzyme	Buffer	BSA	MilliQ	total
2 cuts	（°°） µL	µL	µL	µL	µL	µL	µL
NC	µL	µL	µL	µL	µL	µL	µL

Liquid Culture

No name

Sample	medium
Pcon-pT181anntisense-spinach-DT(4k5)	-

Ligation

Nakamoto and Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
	0.5	2.5	2.5	1.5	0.75	0.75		25

PreDenature	Denature	Annealing	Extension	cycle
°C	°C	°C	°C	--
（◎Д◎）

Transformation

Tatsui

Name	Sample	Competent Cells	Total	Plate
aptamer12-1R-DT	µL	µL	µL	CP
pSB1C3(EcoRI+Spel)+pT181attenuator(EcoRI+Spal)	µL	µL	µL	CP
Pcon-pT181attenuator(EcoRI+Xbal)+RBS-GFP-DT(EcoRI+Spel)	µL	µL	µL	Amp
Pcon-RBS-GFP-DT(EcoRI+Xbal)+Pcon-RBS-tetR-DT(EcoRI+Spel)	µL	µL	µL	Amp
pSB1C3(EcoRI+Spel)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+Spel)	µL	µL	µL	CP

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder
2	DT(2-1)

File:Igku xxxxxx.xxx

精製？

Restriction Enzyme Digestion

	DNA	restricion enzyme	restriction enzyme	Buffer	BSA	MilliQ	total
2 cuts	（°°） µL	µL	µL	µL	µL	µL	µL
NC	µL	µL	µL	µL	µL	µL	µL

液体培養

プレーティング

Colony PCR

No name

Sample	base pair
(｀・ω・´)

PreDenature	Denature	Annealing	Extension	cycle
°C	°C	°C	°C	--
（◎Д◎）

RNA抽出

cDNA合成

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder
2	Pcon-RBS-GFP-DT(RNA)

File:Igku xxxxxx.xxx

Ligation

じっけんしゃ

state	Vector		Inserter		Ligation High ver.2
experiment	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL

Template:Kyoto/Notebook/Sep 25

From 2013.igem.org

Contents

Sep 25

Colony PCR

Electrophoresis

Transformation

Ligation

Digestion

Liquid Culture

Gel Extraction

Restriction Enzyme Digestion

Liquid Culture

Ligation

PCR

Transformation

Electrophoresis

精製？

Restriction Enzyme Digestion

液体培養

プレーティング

Colony PCR

RNA抽出

cDNA合成

Electrophoresis

Ligation