Template:Kyoto/Notebook/Sep 7

From 2013.igem.org

Sep 7

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
RBS-lysis1-DT	195.3
RBS-lysis3-DT	255.5	1.91	2.04
Ptet-pT18	183.6	1.85	1.48
Pcon-RBS-tetR-DT	134.3	1.94	2.20
Ptet-RBS-lacZα-DT	67.7	1.92	1.79
Pcon-pT181 attenuator	232.3	1.95	1.90
pSB4K5	237.0	1.61	1.50
pT181 antisense(pSB1C3)	256.4	1.63	1.78
apt12_1R(pSB1C3)	230.0	1.98	2.28

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Plac-Spinach-DT -5
3	Plac-Spinach-DT -6
4	Plac-Spinach-DT -7
5	Plac-Spinach-DT -8
6	Plac-Spinach-DT -9
7	Plac-Spinach-DT -10
8	100bp ladder
1	100bp ladder
2	Plac-Spinach-DT -11
3	Plac-Spinach-DT -12
4	Pcon-antisense -5
5	Pcon-antisense -6
6	Pcon-antisense -7
7	Pcon-antisense -8
8	100bp ladder

Lane	Sample
1	100bp ladder
2	Pcon-antisense -9
3	Pcon-antisense -10
4	Pcon-antisense -11
5	Pcon-antisense -12
6	Plac-attenuator -5
7	Plac-attenuator -6
8	100bp ladder
1	100bp ladder
2	Plac-attenuator -7
3	Plac-attenuator -8
4	Plac-attenuator -9
5	Plac-attenuator -10
6	Plac-attenuator -11
7	Plac-attenuator -12
8	100bp ladder

Lane	Sample
1	100bp ladder
2	Plac-pT181 antisense

Colony PCR

No name

Sample
Plac-spinach-DT -13~20
Plac-pT181 attenuator -13~20

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68°C	--
5 min	30 s	30 s	42 sec	30 cycles

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/6 Plux (SpeI&PstI)	2 µL	9/7 RBS-lysis2-DT (EcoRI&XbaI)	µL	3.5 µL
experiment	9/6 Plac (SpeI&PstI)	µL	9/7 RBS-lysis2-DT (EcoRI&XbaI)	µL	3.5 µL
experiment	9/6 Pcon-RBS-luxR-DT (EcoRI&XbaI)	1.9 µL	9/6 Plux-RBS-GFP-DT(SpeI&PstI)	2.2 µL	3.5 µL
experiment	9/6 Pbed/araC (SpeI&PstI)	1.6 µL	9/5 RBS-luxI-DT (EcoRI&XbaI)	16 µL	3.5 µL
experiment	9/6 Plac (SpeI&PstI)	1.5 µL	9/5 pT181 antisense (EcoRI&XbaI)	2.0 µL	3.5 µL
experiment	9/6 Pcon (SpeI&PstI)	3.0 µL	9/5 RBS-luxI-DT (EcoRI&XbaI)	10 µL	3.5 µL

Electrophoresis

Nakamoto

Lane	Sample
1	100bp ladder
2	Plac-Spinach-DT -13
3	Plac-Spinach-DT -14
4	Plac-Spinach-DT -15
5	Plac-Spinach-DT -16
6	Plac-Spinach-DT -17
7	Plac-Spinach-DT -18
8	Plac-Spinach-DT -19
9	Plac-Spinach-DT -20
10	Plac-pT181 attenuator -13
11	Plac-pT181 attenuator -14
12	100bp ladder

Lane	Sample
1	100bp ladder
2	Plac-pT181 attenuator -15
3	Plac-pT181 attenuator -16
4	Plac-pT181 attenuator -17
5	Plac-pT181 attenuator -18
6	Plac-pT181 attenuator -19
7	Plac-pT181 attenuator -20
8	100bp ladder

Transformation

Tatsui

Name	Sample	Competent Cells	Total
Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT	2µL	20µL	22µL
Pbed/araC+RBS-luxI-DT	2µL	20µL	22µL
Plac+pT181 antisense	2µL	20µL	22µL
Pcon+RBS-luxI-DT	2µL	20µL	22µL

Restriction Enzyme Digestion

No Name

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/7 RBS-lysis1-DT(XbaI & PstI)	10 µL	1 µL	1 µL	3 µL	3 µL	12 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/7 RBS-lysis3-DT (XbaI & PstI)	7.8 µL	1 µL	1 µL	3 µL	3 µL	14.2 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
9/5 Plac (SpeI & PstI)	6.0 µL	1 µL	1 µL	3 µL	3 µL	16.0 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	EcorI	SpeI	Buffer	BSA	MilliQ	total
9/7 pSB4K5 (EcoRI & SpeI)	8.4 µL	1 µL	1 µL	3 µL	3 µL	16.0 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	restricion enzyme	restriction enzyme	Buffer	BSA	MilliQ	total
Pcon-pT181 attenuator (SpeI & PstI)	8.6 µL	1 µL	1 µL	3 µL	3 µL	13.4 µL	30 µL
Pcon-pT181 attenuator (EcorI & SpeI)	8.6 µL	1 µL	1 µL	3 µL	3 µL	13.4 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/5 Spinach-DT (XbaI & PstI)	5.4 µL	1 µL	1 µL	3 µL	3 µL	16.6 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
Ptet-anticodone (SpeI & PstI)	11 µL	1 µL	1 µL	3 µL	3 µL	11 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/6 RBS-lacZα-DT (XbaI & PstI)	11 µL	1 µL	1 µL	3 µL	3 µL	11 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	EcoRI	SpeI	Buffer	BSA	MilliQ	total
pSB1C3 (1) (EcoRI & SpeI)	6.6 µL	1 µL	1 µL	3 µL	3 µL	15.4 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
pSB1C3 (3) (XbaI & PstI)	6.6 µL	1 µL	1 µL	3 µL	3 µL	15.4 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	RBS-lysis1-DT (9/7)	XbaI	PstI
3	RBS-lysis1-DT (9/7)	--	--
4	RBS-lysis3-DT (9/7)	XbaI	PstI
5	RBS-lysis3-DT (9/7)	--	--
6	Pcon-pT181 attenuator	EcoRI	SpeI
7	Pcon-pT181 attenuator	--	--
8	9/5 Spinach-DT	XbaI	PstI
9	9/5 Spinach-DT	--	--
10	100bp ladder	--	--
11	100bp ladder	--	--
12	RBS-lacZα-DT	XbaI	PstI
13	RBS-lacZα-DT	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Ptet-antisense	SpeI	PstI
3	Plac (NC)	--	--
4	pSB4K5	EcoRI	SpeI
5	pSB4K5	--	--
6	Pcon-pT181 attenuator	SpeI	PstI
7	1kbp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Plac	SpeI	PstI
3	Ptet-antisense	--	--
4	pSB1C3	EcoRI	SpeI
5	pSB1C3	--	--
6	pSB1C3	XbaI	PstI
7	pSB1C3	--	--
8	1kbp ladder	--	--

Liquid Culture

No name

Sample	medium
entA- strain	SOB medium (+Kan)
9/15 Pcon-pT181 antisense	Plusgrow (+Amp)

Template:Kyoto/Notebook/Sep 7

From 2013.igem.org

Contents

Sep 7

Miniprep

Electrophoresis

Colony PCR

Ligation

Electrophoresis

Transformation

Restriction Enzyme Digestion

Electrophoresis

Liquid Culture