Template:Kyoto/Notebook/Sep 24

From 2013.igem.org

(Difference between revisions)

Revision as of 12:31, 26 September 2013

Sep 24

Electrophoresis

No name

Lane	Sample
1	9/20 Pcon-pT181aptamer12-1R-DT 1
2	9/20 Pcon-pT181aptamer12-1R-DT 2
3	9/20 Pcon-pT181aptamer12-1R-DT 3
4	9/20 Pcon-pT181aptamer12-1R-DT 4
5	9/20 Pcon-pT181aptamer12-1R-DT 5
6	9/20 Pcon-pT181aptamer12-1R-DT 6
7	9/20 Pcon-pT181aptamer12-1R-DT 7
8	9/20 Pcon-pT181aptamer12-1R-DT 8
9	100bp ladder
10	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
11	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
12	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
13	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
14	9/20 Plac-aptamer12-1R-DT 1
15	9/20 Plac-aptamer12-1R-DT 2
16	9/20 Plac-aptamer12-1R-DT 3
17	9/20 Plac-aptamer12-1R-DT 4

File:Igkuxxxx.png

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 pSB1C3-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
9/23 Plac-+spinach-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
PUC19	2 µL	20 µL	22 µL	LB (+Amp)
Ptet-RBS-lacZα-DT	2 µL	20 µL	22 µL	LB (+CP)
pSB1C3	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
aptamer12-1R-DT(Master plate)	LB(+CP)

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 Pcon-attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 pT181attenuator(1C3)	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
pSB1C3 (Master plate-1)	LB(+CP)

Digestion

Kojima

	8/21 pT181attenuator (330 µg/mL)	XbaI	PstI	buffer	BSA	MilliQ	total
2 cuts	6.1	1	1	3	3	15.9	30
NC	0.3	0	0	1	1	7.7	10

	9/22 Pcon-pT181attenuator (368.3 µg/mL)	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	5.4	1	1	3	0	19.6	30
NC	0.3	0	0	1	0	8.7	10

	9/16 pSB1C3	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	18	1	1	3	0	7.0	30
NC	0.9	0	0	1	0	8.1	10

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	-	-
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	--	--
4	Pcon-pT181attenuator	EcoRI	SpeI
5	Pcon-pT181attenuator	--	--
6	pSB1C3	EcoRI	SpeI
7	pSB1C3	--	--

File:Xxx

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/24 aptamer12-1R-DT	144.7	1.03	1.21

Gel Extraction

Kojima

Lane	DNA	Enzyme1	Enzyme2
1	100kb ladder	--	--
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	XbaI	PstI
4	pT181attenuator	XbaI	PstI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	SpeI
7	Pcon-pT181attenuator	EcoRI	SpeI
8	Pcon-pT181attenuator	EcoRI	SpeI
9	--	--	--
10	pSB1C3	EcoRI	SpeI
11	pSB1C3	EcoRI	SpeI
12	pSB1C3	EcoRI	SpeI

File:Igkuxxx.jpg
File:Igku Axxxjpg

Name	concentration[µg/mL]	260/280	260/230
pT181attenuator(XbaI&PstI)	10.1	2.02	0.43
Pcon-pT181attenuator(EcoRI&SpeI)	10.3	1.81	0.32
pSB1C3(EcoRI&SpeI)	10.0	2.81	0.02

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8	1414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8	1631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4	2042
9/23 Pcon-pT181antisense-DT 1	508
9/23 Pcon-pT181antisense-DT 2	508
9/23 Pcon-pT181antisense-DT 3	508
9/23 Pcon-pT181antisense-DT 4	508
9/23 Pcon-pT181antisense-DT 5	508
9/23 Pcon-pT181antisense-DT 6	508
9/23 Pcon-pT181antisense-DT 7	508
9/23 Pcon-pT181antisense-DT 8	508

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 sec	30 sec	2min	30 cycles

Lane	Sample
1	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
6	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
7	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
8	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
9	1kb ladder
10	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
11	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
12	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
13	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
14	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
15	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
16	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
17	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

File:Igkuxxxx.png
</div>

Lane	Sample
1	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-pT181antisense-DT 1
6	9/23 Pcon-pT181antisense-DT 2
7	1kb ladder
8	9/23 Pcon-pT181antisense-DT 3
9	9/23 Pcon-pT181antisense-DT 4
10	9/23 Pcon-pT181antisense-DT 5
11	9/23 Pcon-pT181antisense-DT 6
12	9/23 Pcon-pT181antisense-DT 7
13	9/23 Pcon-pT181antisense-DT 8
14	--

File:Igkuxxxx.png
</div>

Restriction Enzyme Digestion

Nakamoto

	9/24 aptamer12-1R-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	17.5µL	1.0µL	1.0µL	3.0µL	3.0µL	4.5µL	30µL
NC	0.9µL	0µL	0µL	1.0µL	1.0µL	7.1µL	10µL

9/20 Ptet	SpeI	PstI	buffer	BSA	MilliQ	total
2cuts	9.5µL	1.0µL	1.0µL	3.0µL	0µL	15.5µL	30µL
NC	0.5µL	0µL	0µL	1.0µL	0µL	8.5µL	10µL

9/22 RBS-GFP-DT	EcoRI	SpeI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	0µL	21.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	0µL	8.8µL	10µL

9/22 Pcon-RBS-GFP-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	2.4µL	1.0µL	1.0µL	3.0µL	3.0µL	19.6µL	30µL
NC	0.1µL	0µL	0µL	1.0µL	1.0µL	7.9µL	10µL

9/22 Pcon-pT181attenuator 1	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	5.4µL	1.0µL	1.0µL	3.0µL	3.0µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µL	10µL

9/22 RBS-GFP-DT	XbaI	PstI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	3.0µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	1.0µL	7.8µL	10µL

Ligation

Nakamoto

state	Vector		Inserter		Ligation High ver.2
	9/22 aptamer12-1R-DT(EcoRI&XbaI)	1.4 µL	9/24 Pcon-pT181attenuator(EcoRI&SpeI)	26 µL	3.5 µL
	9/4 Ptet(SpeI&PstI)	2.0 µL	9/21 RBS-GFP-DT(XbaI&PstI)	9.0 µL	3.5 µL

incubated at 37 °C for 1 hour.

colony PCR

Kojima

Sample	base pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12	2042bp
9/23 Pcon-pT181antisense-DT 9	508bp
9/23 Pcon-pT181antisense-DT 10	508bp
9/23 Pcon-pT181antisense-DT 11	508bp
9/23 Pcon-pT181antisense-DT 12	508bp
9/23 Pcon-pT181antisense-DT 13	508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 1	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 2	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 3	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 4	1527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4	859bp
9/24 pT181attenuator(pSB1C3) 1	390bp
9/24 pT181attenuator(pSB1C3) 2	390bp
9/24 pT181attenuator(pSB1C3) 3	390bp
9/24 pT181attenuator(pSB1C3) 4	390bp

Tatsui

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycle

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
pSB1C3	183.9	1.91	1.59
Pcon-pT181attenuator-aptamer12-1R-DT	231.7	1.82	2.35
Plac-aptamer12-1R-DT	208.9	1.98	1.79

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1kb ladder	--	--
2	9/24 aptamer12-1R-DT	EcoRI	XbaI
3	9/24 aptamer12-1R-DT	--	--
4	9/24 Ptet	SpeI	PstI
5	9/24 Ptet	--	--
6	9/24 RBS-GFP-DT	EcoRI	SpeI
9/24 RBS-GFP-DT	--	--
8	9/24 Pcon-RBS-GFP-DT	EcoRI	XbaI
8	9/24 Pcon-RBS-GFP-DT	--	--
9	9/24 Pcon-RBS-GFP-DT	--	--
10	9/24 Pcon-pT181attenuator 1	EcoRI	XbaI
11	9/24 Pcon-pT181attenuator 1	--	--
12	9/24 RBS-GFP-DT	XbaI	PstI
13	9/24 RBS-GFP-DT	--	--
14	1kb ladder

File:Igkuxxxx.jpg

Gel Extraction

No name 1

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	aptamer12-1R-DT	EcoRI	XbaI
3	aptamer12-1R-DT	EcoRI	XbaI
4	aptamer12-1R-DT	EcoRI	XbaI
5	--	--	--
6	Ptet	SpeI	PstI
7	Ptet	SpeI	PstI
8	Ptet	SpeI	PstI
9	--	--	--
10	RBS-GFP-DT	EcoRI	SpeI
11	RBS-GFP-DT	EcoRI	SpeI
12	RBS-GFP-DT	EcoRI	SpeI

File:Igku xxxbefore.jpg
File:Igkuxxx1-2.jpg

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	Pcon-RBS-GFP-DT	EcoRI	XbaI
3	Pcon-RBS-GFP-DT	EcoRI	XbaI
4	Pcon-RBS-GFP-DT	EcoRI	XbaI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	XbaI
7	Pcon-pT181attenuator	EcoRI	XbaI
8	Pcon-pT181attenuator	EcoRI	XbaI
9	--	--	--
10	RBS-GFP-DT	XbaI	PstI
11	RBS-GFP-DT	XbaI	PstI
12	RBS-GFP-DT	XbaI	PstI

File:Igkuxxxxpg
File:Igkuxxxx2.jpg

Electrophoresis

No name
1

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
8	1kb ladder

File:Igkuxxxx.jpg

2

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4	Pcon-pT181attenuator-RBS-GFP-DT 1
5	Pcon-pT181attenuator-RBS-GFP-DT 2
6	Pcon-pT181attenuator-RBS-GFP-DT 3
7	Pcon-pT181attenuator-RBS-GFP-DT 4
8	1kb ladder

File:Igkuxxxx.jpg

3

Lane	Sample
1	Pcon-pT181antisense-DT 9
2	Pcon-pT181antisense-DT 10
3	Pcon-pT181antisense-DT 11
4	Pcon-pT181antisense-DT 12
5	Pcon-pT181antisense-DT 13
6	100bp ladder
7	Pcon-pT181attenuator-aptamer12-1R-DT 1
8	Pcon-pT181attenuator-aptamer12-1R-DT 2

File:Igkuxxxx.jpg

4

Lane	Sample
1	Pcon-pT181attenuator-aptamer12-1R-DT 3
2	Pcon-pT181attenuator-aptamer12-1R-DT 4
3	100bp ladder
4	pT181attenuator(pSB1C3) 1
5	pT181attenuator(pSB1C3) 2
6	pT181attenuator(pSB1C3) 3
7	pT181attenuator(pSB1C3) 4

File:Igkuxxxx.jpg

@@ Line 363: / Line 363: @@
 <span class="author">Nakamoto</span>
 {| class="wikitable"
-!9/24 aptamer12-1R-DT ||EcoRI||XbaI || buffer||BSA||MilliQ||total
+!||9/24 aptamer12-1R-DT ||EcoRI||XbaI || buffer||BSA||MilliQ||total
 |-
 |2cuts||17.5&micro;L||1.0&micro;L ||1.0&micro;L ||3.0&micro;L ||3.0&micro;L ||4.5&micro;L ||30&micro;L
@@ Line 403: / Line 403: @@
 |-
 |NC||0.2&micro;L ||0&micro;L ||0&micro;L ||1.0&micro;L ||1.0&micro;L ||7.8&micro;L ||10&micro;L
 |}
 ===Ligation===

Template:Kyoto/Notebook/Sep 24

From 2013.igem.org

Revision as of 12:31, 26 September 2013

Contents

Sep 24

Electrophoresis

Transformation

Liquid Culture

Transformation

Liquid Culture

Digestion

Electrophoresis

Miniprep

Gel Extraction

Colony PCR

Restriction Enzyme Digestion

Ligation

colony PCR

Miniprep

Electrophoresis

Gel Extraction

Electrophoresis