Template:Kyoto/Notebook/Sep 13

From 2013.igem.org

Sep 13

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
9/12 Fusion6 antisense	245.9	1.88	1.41
9/12 Plux-3	115.5	1.58	1.32
9/12 Plux-4	125.3	1.93	1.44
9/12 Fusion3n2 attenuator-1	231.9	1.81	1.31

Gel Extraction

Tatsui,Ashida

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lysis1-DT	XbaI&PstI
3	RBS-lysis1-DT	XbaI&PstI
4	RBS-lysis1-DT	XbaI&PstI

Lane	DNA	Enzyme
1	1kb ladder	--
3	RBS-lysis2-DT	XbaI&PstI
4	RBS-lysis2-DT	XbaI&PstI
5	RBS-lysis2-DT	XbaI&PstI

Lane	DNA	Enzyme
3	RBS-lysis3-DT	XbaI&PstI
4	RBS-lysis3-DT	XbaI&PstI
5	RBS-lysis3-DT	XbaI&PstI
6	1kb ladder	--

Lane	DNA	Enzyme
2	1kb ladder	--
3	pSB1C3	EcoRI&SpeI
4	pSB1C3	EcoRI&SpeI
5	pSB1C3	EcoRI&SpeI

Lane	DNA	Enzyme
1	1kb ladder	--
2	pSB1C3	XbaI&PstI
3	pSB1C3	XbaI&PstI
4	pSB1C3	XbaI&PstI

Restriction Enzyme Digestion

Nakamoto

	9/13 Plac	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	16	0µL	0µL	0µL	1µL	3µL	3µL	7µL	30µL
NC	0.8	0µL	0µL	0µL	0µL	1µL	1µL	7.2µL	10µL

	9/10 Pcon attenuator	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(PstI)	10	0µL	0µL	0µL	1µL	3µL	3µL	13µL	30µL
NC	0.5	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/12 Plux	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	12	0µL	0µL	0µL	1µL	3µL	3µL	11µL	30µL
NC	0.6	0µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/12 DT-E	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(XbaI)	23.7	0µL	0µL	1µL	0µL	3µL	3µL	0µL	30.7µL
NC	1.2	0µL	0µL	0µL	0µL	1µL	1µL	6.8µL	10µL

	9/12 Pcon-RBS-luxR-bTE	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(XbaI)	22.4	0µL	0µL	1µL	0µL	3µL	3µL	0.6µL	30µL
NC	2.2	0µL	0µL	0µL	0µL	1µL	1µL	5.8µL	10µL

	9/12 Pcon-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(SpeI)	21	0µL	1µL	0µL	0µL	3µL	3µL	12.0µL	30µL
NC	2.1	0µL	0µL	0µL	0µL	1µL	1µL	5.7µL	10µL

M9 (EDTA)

Kanbayashi

1mM EDTA

volume	10ml
Na2HPO4	300mg
KH2PO4	150mg
NaCl	25mg
NH4Cl	50mg
Fe(3)-EDTA	21mg

5mM EDTA

volume	10ml
Na2HPO4	300mg
KH2PO4	150mg
NaCl	25mg
NH4Cl	50mg
Fe(3)-EDTA	105mg

</div>

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	1kb ladder	--
3	Pcon-P	SpeI
4	Pcon-P	SpeI
5	Pcon-P	SpeI

Liquid Culture

Tatsui

Sample	medium
9/11 aptamer12_1M(pSB1C3)-1	--
9/11 aptamer 12_P1	--
9/11 Fusaion1 antisense-4	--
9/12 pSB4K5-1	--
9/12 pSB4K5-2	--

Restriction Enzyme Digestion

Nakamoto

	9/13 Plac-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(SpeI)	23.5	0µL	0.5µL	0µL	0µL	3µL	3µL	0µL	30µL
NC	8	0µL	0µL	0µL	0µL	1µL	1µL	0µL	10µL

	9/13 Pcon-pT181 attenuator-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(SpeI)	23.5	0µL	0.5µL	0µL	0µL	3µL	3µL	0µL	30µL
NC	02.3	0µL	0µL	0µL	0µL	1µL	1µL	5.7µL	10µL

	9/13 Plux-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(SpeI)	23.8	0µL	0.2µL	0µL	1µL	3µL	3µL	0µL	30µL
NC	8	0µL	0µL	0µL	0µL	1µL	1µL	0µL	10µL

	9/5 RBS-lacZα-DT	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cut(XbaI&PstI)	10.8	0µL	0µL	1µL	1µL	3µL	3µL	11.2µL	30µL
NC	0.5	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Electrophoresis

Hirano

Lane	Sample
1	100bp ladder
2	RBS-lacZα-DT(XbaI&PstI)
3	RBS-lacZα-DT(NC)

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Plac (SpeI&PstI)	8.5µL	9/13 pT181 antisense (XbaI&PstI)	2.1 µL	5.3 µL
experiment	9/13 Plac (SpeI&PstI)	8.5 µL	9/10 aptamer 12_1R-DT (XbaI&PstI)	1.8 µL	5.15µL
experiment	9/13 Plac (SpeI&PstI)	8.5 µL	9/2 pT181 attenuator(XbaI&PstI)	2.3 µL	10.4 µL
experiment	9/13 Plux (SpeI&PstI)	11.4 µL	9/13 RBS-lysis1-DT (XbaI&PstI)	5.0 µL	8.2 µL
experiment	9/13 Plux (SpeI&PstI)	11.4 µL	9/13 RBS-lysis2-DT (XbaI&PstI)	7.0 µL	9.2 µL
experiment	9/13 Plux (SpeI&PstI)	7.2 µL	9/13 RBS-lysis3-DT (XbaI&PstI)	6.0 µL	6.6 µL
experiment	9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)	2.0 µL	9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)	5.2 µL	3.6 µL
experiment	9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)	2.0 µL	9/10 aptamer 12_1R-DT (XbaI&PstI)	1.6 µL	1.8 µL
experiment	9/12 Ptet-pT181 antisense (--)	1.0 µL	9/10 spinach-DT (--)	2.4 µL	1.7 µL

PCR

No name

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/7 tetR aptamer 12_1R(pSB1C3)	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/6 pT181 antisense-1(pSB1C3)	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/6 pT181 attenuator (pSB1C3)	0.5	3.85	1.75	3.9	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/27 Pλ-RBS-luxI-DT-1	0.5	2.55	1.75	5.2	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/31 Pbad/araC-RBS-RFP-2	0.5	3.75	1.75	4	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/13 Plac(pSB1A2)	0.5	4.2	1.75	3.5	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/20 J23100-RBS-lacZα-DT	0.5	6.35	1.75	1.4	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/7 Ptet-RBS-lacZα-DT	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/8 J23100-spinach-DT	0.5	5.75	1.75	2	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/11 RBS-lysis1-DT	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/12 RBS-lysis2-DT	0.5	6.75	1.75	1.0	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/12 RBS-lysis3-DT	0.5	6.75	1.75	1.0	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/8 J23100-spinach-DT	0.5	6.35	1.75	1.4	0	0.5	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/27 Pλ-RBS-luxI-DT-1	0.5	2.55	1.75	5.2	0	0.5	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/12 RBS-lysis2-DT	0.5	6.75	1.75	1.0	0	0.5	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/11 RBS-lysis1-DT	0.5	6.05	1.75	1.7	0	0.5	10.5

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	68°C	--
2m	10s	5s	4m	30

PCR

Yoshida

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	Prefix-RBS-RpaB_fwd primer	RpaB-GG4-suffix_rev primer	MilliQ	total
RpaB	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	Prefix-PkaiBC_fwd primer ver.3	PkaiBC-suffix_rev primer ver.3	MilliQ	total
RpaB	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58°C	68°C	--
2m	10s	30s	30s	30

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

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/13 Plac+pT181 antisense	2µL	20µL	22µL	Amp
9/13 Plac+apt12_1R	2µL	20µL	22µL	Amp
9/13 Plac+pT181 attenuator	2µL	20µL	22µL	Amp
9/13 Plux+pT181 antisense	2µL	20µL	22µL	CP
9/13 Plux+RBS-lysis2-DT	2µL	20µL	22µL	CP
9/13 Plux+RBS-lysis2-DT	2µL	20µL	22µL	CP
9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT	2µL	20µL	22µL	Amp
9/13 Pcon-pT181 antisense+apt12_1R-DT	2µL	20µL	22µL	Amp
9/13 Ptet-pT181antisense+spinach-DT	2µL	20µL	22µL	CP
Pλ-luxI-1	2µL	20µL	22µL	Amp

Template:Kyoto/Notebook/Sep 13

From 2013.igem.org

Contents

Sep 13

Miniprep

Gel Extraction

Restriction Enzyme Digestion

M9 (EDTA)

Gel Extraction

Liquid Culture

Restriction Enzyme Digestion

Electrophoresis

Ligation

PCR

PCR

Gel Extraction

Transformation