Template:Kyoto/Notebook/Sep 27

From 2013.igem.org

Sep 27

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI+SpeI
3
4
6	Pcon-attenuator-aptmer-DT	EcoRI+SpeI
7
8
10	Pcon-attenuator-aptmer-DT	EcoRI+SpeI
11
12

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator-DT(EcoRI+SpeI)	6.8	1.98	0.43
pT181-attenuator(EcoRI+SpeI)	7.5	1.84	0.06
pSB1C3(EcoRI+SpeI)	17.7	1.97	0.71

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	pSB4K5	EcoRI+PstI
4	pSB4K5	EcoRI+PstI
5	pSB4K5	EcoRI+PstI
7	pSB6A1	EcoRI+PstI
8	pSB6A1	EcoRI+PstI
9	pSB6A1	EcoRI+PstI

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	pSB4K5	EcoRI	PstI
2	pSB4K5	-	-
3	1kbp ladder	-	-
4	Pcon-attenuator	EcoRI	SpeI
5	Pcon-attenuator	-	-

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT	XbaI	PstI
2	RBS-GFP-DT	-	-
3	RBS-GFP-DT	EcoRI	XbaI
4	1kbp ladder	-	-
5	Ptet	EcoRI	SpeI
6	Ptet	-	-

Liquid Culture

No name

Sample	medium
Pcon-GFP	Amp
RBS-GFP-DT	CP
Pcon-attenuator	Amp
Pcon-attenuator-aptamer-DT	Amp
Pcon-tetR-DT	Amp

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL	10µL	9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL	1.3µL	3.5 µL
experiment	9/25 DT (EcoRI+XbaI) 42.8ng/µL	2.3µL	9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL	1.5µL	1.9 µL
experiment	9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL	7.8µL	9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL	8.0µL	3.5 µL
experiment	9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL	7.8µL	9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL	12µL	3.5 µL
experiment	9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL	5.7 µL	8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL	12.6µL	3.5 µL

Liquid Culture

Hirano

Sample	medium
Pcon-spinach-DT	plusgrow Amp
Pcon-tetRaptamer-DT	plusgrow Amp
Pcon-antisense-spinach-DT	plusgrow Amp
spinach-DT	plusgrow CP

RNA Extraction

Nakamoto

Name	concentration[µg/mL]	260/280	260/230
9/26 Pcon-GFP-DT	423.0	1.83	1.46
9/26 Pcon-GFP-DT K	298.7	1.82	1.50

Electrophoresis

No name

Lane	Sample
1	9/27 Pcon-GFP-DT(RNA)
2	1kb ladder
3	9/27 Pcon-GFP-DT K(RNA)

Miniprep

Tatsui

DNA
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-antisense
Pcon-tetR-DT
Pcon-attenuator-DT
Pcon-antisense-spinach-DT

Transformation

Hirano

Name1	Name2	Sample1(µL)	Sample2(µL)	Competent Cells(µL)	Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-attenuator-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-antisense-spinach-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-tetR aptamer-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-attenuator-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-antisense-spinach-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-tetR aptamer-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	--	2	--	20	22
Pcon-RBS-GFP-DT(4K5)	--	2	--	20	22
attenuator(1C3)	--	2	--	20	22

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pcon-RBS－GFP-DT	137.6	1.95	1.78
RBS-GFP-DT	77.0	2.06	1.96
Pcon-PT181 attenuator	231.0	1.89	1.89
Pcon-antisense-Spinach-DT(2)	130.0	1.56	1.62
Pcon-aptamer-DT	218.8	1.92	1.72
Pcon-attenuator-DT	180.7	1.95	1.78
Pcon-Spinach-DT	177.6	1.96	1.06
Pcon-antisense-Spinach-DT(1)	181.1	1.96	1.92
Pcon-antisense	152.9	2.02	1.96
Pcon-RBS+ezR-DT	164.1	1.93	1.91

Restriction Enzyme Digestion

No name

	9/27 RBS-GFP-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	12µL	1µL	1µL	3µL	3µL	20µL	40µL
NC	1.3µL	0µL	0µL	1µL	1µL	6.7µL	10µL

	9/27 Pcon-tetR-DT	EcoRI	PstI	Buffer	MilliQ	total
2 cuts	12.2µL	1µL	1µL	3µL	12.8µL	30µL
NC	0.6µL	0µL	0µL	1µL	8.4µL	10µL

RNA Extraction

Nakamoto

Name	concentration[µg/mL]	260/280	260/230
9/27 Pcon-RBS-GFP-DT	686.6	1.99	1.34
9/27 Pcon-RBS-GFP-DT (+High salt)	127.6	1.76	0.27

DNA sythesis

No name

genome DNA	DNA volume	genome DNA wipeout buffer	RNase freewater	RT	RT buffer	Primer Mix	total
9/27 Pcon-RBS-GFP-DT	0.8µL	1µL	5.2µL	0.5µL	2µL	0.5µL	10µL
9/27 pcon-RBS-GFP-DT(+high salt)	2.5µL	1µL	3.5µL	0.5µL	2µL	0.5µL	10µL
9/27 Pcon-RBS-GFP-DT(revised concentration)	0.5µL	1µL	5.5µL	0.5µL	2µL	0.5µL	10µL

Electrophoresis

No name

Lane	Sample
1	1kb ladder
2	Pcon-RBS-GFP-DT
3	Pcon-RBS-GFP-DT

Liquid Culture

No name

Sample	medium
Pcon spinach DT	plusgrow 2µL(Amp)
Pcon aptamer DT	plusgrow 2µL(Amp)
Pcon antisense Spinach DT	plusgrow 2µL(Amp)
Spinach DT	plusgrow 4µL(CP)

RT PCR

No name

genome DNA	KOD plus	10x buffer for KOD-plus-ver.2	2nM dNTPs	25mM MgSO4	F primer	R primer	MilliQ	total
0.5	1	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	49.8°C	68°C	--
2min	10sec	30sec	10sec	30

Electrophoresis

No name

Lane	Sample
1	Pcon-GFP-DT(GFP)
2	Pcon-GFP-DT(internal)
3	Pcon-GFP-DT(HS)(GFP)
4	Pcon-GFP-DT(internal)

Lane	Sample
1	Pcon-GFP-DT(GFP)
2	Pcon-GFP-DT(internal)
3	100bp ladder

Digesting cut check

No name

Lane	Sample
1	RBS-GFP-DT(XbaI&PstI)
2	RBS-GFP-DT NC(XbaI&PstI)
3	1kbp ladder
4	Pcon-RBS-tetR-DT(EcoRI&SpeI)
5	Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	RBS-GFP-DT	Xbal+PstI
4	RBS-GFP-DT	Xbal+PstI
5	RBS-GFP-DT	Xbal+PstI
7	1kbp ladder	--
9	Pcon-tetR-DT	EcoRI+SpeI
10	Pcon-tetR-DT	EcoRI+SpeI
11	Pcon-tetR-DT	EcoRI+SpeI

RNA Extraction

Nakamoto

	0	5min	15min	20min	23min	28min	33min	36min
aptamer generator	0.348	0.408	0.550	0.865
Pcon-spinach-DT	0.250	0.240	0.305	0.426	0.471	0.554	0.668	0
GFP generator	0.389	0.420	0.516	0.884
antisense-spinach	0.371	0.438	0.537	0.733
Spinach-DT	0.084	0.110	0.134	0.433	0.269	0.326	0.409	0.742

Name	concentration[µg/mL]	260/280	260/230
Spinach-DT	83.9	1.63	0.17
Pcon-tetRaptamer-DT	425.1	2.10	0.64
Pcon-RBS-GFP-DT	262.4	2.01	0.84
Pcon-antisense-Spinach-DT	489.1	2.05	0.68
Pcon-Spinach-DT	266.6	2.01	0.66

cDNA Synthesis

Nakamoto

	9/27 Spinach-DT	9/23 Pcon-tetRaptamer-DT	9/23 Pcon-RBS-GFP-DT	9/23 Pcon-antisense-spinach-DT	9/23 Pcon-Spinach-DT
RNA	1	1	1	1	1
gDNA wipeout	6	1.2	1.9	1.0	1.9
RNase-free water	0	4.8	4.1	5.0	4.1
RT	0.5	0.5	0.5	0.5	0.5
RT Buffer	2	2	2	2	2
Primer mix	0.5	0.5	0.5	0.5	0.5
total	10µL	10µL	10µL	10µL	10µL

Template:Kyoto/Notebook/Sep 27

From 2013.igem.org

Contents

Sep 27

Gel Extraction

Gel Extraction

Electrophoresis

Liquid Culture

Ligation

Liquid Culture

RNA Extraction

Electrophoresis

Miniprep

Transformation

Miniprep

Restriction Enzyme Digestion

RNA Extraction

DNA sythesis

Electrophoresis

Liquid Culture

RT PCR

Electrophoresis

Digesting cut check

Gel Extraction

RNA Extraction

cDNA Synthesis