Template:Kyoto/Notebook/Sep 23

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Sep 23

Transformation

No name

Name	Sample	Competent Cells	Plate
9/14 pSB1C3＋PkaiBC	2 µL	20 µL	--
9/14 pSB1C3＋RpaB	2 µL	20 µL	--
9/14 pkaiBC＋Rbs-GFP-DT	2 µL	20 µL	--

Gel Extraction

No Name

Lane	DNA	Enzyme
1	1kbp ladder
3-4	Pcon-RBS-tetR-PT

Name	concentration[µg/mL]	260/280	260/230
Pcon-PT181 anti E+S	46.7	1.06	0.93
Pcon-RBS-tetR-DT E+X	119.3	0.99
RBS-8FP-DT E+X	87.7	1.00	0.98
ap42_1R-DT E+X	68.8	1.32	1.03
Pcon-RBS-tetR-DT 1-2 A	203.0	1.66	1.79
Pcon-RBS-tetR-DT 3-2 A	67.7	2.64	1.28
Pcon-RBS-GFP-DT 1-S A	185.6	1.88	2.04
Pcon-RBS-GFP-DT 2-S A	298.6	1.68	1.55
Pcon-tetR-DT E-2 A	202.4	1.57	1.66

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
9/8 Pcon-pT181 anti	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
8/20 Ptet(1)	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
9/16 Plac 2	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94　°C	98　°C	57　°C	68　°C	--
2min	10sec	30sec	20sec	30cycles

Ligation

Yoshida

state	Vector		Inserter		Ligation High ver.2
experiment	9/21 Pcon-RBS-GFP-DT E+X	47.5 µg/mL	9/15 Pcon-RBS-tetR-DT E+S	59 µg/mL	2.3 µg/mL
experiment	9/21 Pcon-RBS-GFP-DT E+X	47.5 µg/mL	9/15 Pcon-apt12_1R-DT E+S	17 µg/mL	2.4 µg/mL
experiment	9/21 Pcon-RBS-GFRDT E+X	2.1 µg/mL	9/15 Pcon-DT181 attenuator-DT	21 µg/mL	3.0 µg/mL
experiment	9/22 apt 12_1R-DT E+X	67.8 µg/mL	9/18 Pcon-DT181 attenuator E+S	5.7 µg/mL	3.5 µg/mL
experiment	9/22RBS-GFP-DT E+X	87.7 µg/mL	9/18 Pcon-DT18 attenuator E+S	6.7 µg/mL	3.5 µg/mL
experiment	9/13 DT E+X	25.4 µg/mL	9/22 Pcon-DT181 antisense E+S	46.8 µg/mL	2.3 µg/mL

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
Pcon-attenuator-DT (E-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-attenuator-DT (2-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-antisense-spinach-DT (1-2A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-antisense-spinach-DT (E-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25

PreDenature	Denature	Annealing	Extension	cycle
94　°C	98　°C	57　°C	68　°C	--
2min	10s	30s	45s	30cycle

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/22 Plac-DT181 attenuator-1	204.0	2.00	1.95
9/22 Plac-DT181 attenuator-2	195.3	1.84	1.62
9/22 Plac-DT181 antisense-1	188.2	1.87	2.15
9/22 Plac-DT181 antisense-2	165.0	1.95	2.66

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1,6	100bp ladder(Promega)	--	--
2	Pcon-attenuator-DT (E-1A)	--	--
3	Pcon-attenuator-DT (2-1A)	--	--
4	Pcon-antisense-spinach-DT (1-2A)	--	--
5	Pcon-antisense-spinach-DT (E-1A)	--	--

Gel Extraction

Hirano

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator-DT(E-1A)	166.7	1.51	0.99
Pcon-pT181 attenuator-DT(2-1A)	122.1	1.75	1.20
Pcon-pT181 antisense-spinach-DT(1-2A)	99.1	1.86	1.81

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	9/8 Pcon-pT181 antisense(E-1A)	--	--
3	8/20 Ptet(1)(2-S)	--	--
4	9/16 Plac2(E-1A)	--	--

Transformation

No Name

Name	Sample	Competent Cells	Total	Plate
pSB1C3	2µL	20µL	22µL

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/19 Plac S+P	24.6 µg/mL	9/18 ape12_1R-DT X+P	4.5 µg/mL	2 µg/mL
experiment	9/21 RBS-GFP-DT E+X	88.4 µg/mL	9/15 Pcon-pT181 attenuator E+S	21 µg/mL	2 µg/mL
experiment	9/13 PSB1C3 X+P	26.0 µg/mL	9/16 pT181 attenuator X+P	8.2 µg/mL	2 µg/mL

Colony PCR

Tatsui

Sample	base pair
9/22 Pcon-PT181 attenuator12-1R-DT(1~8)	--
9/22 Pcon-PT181 attenuator(1~4)	--
9/20 Plac＋aptamer12-1R-DT(1~4)	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	54s	30cycle

Template:Kyoto/Notebook/Sep 23

From 2013.igem.org

Contents

Sep 23

Transformation

Gel Extraction

PCR

Ligation

PCR

Miniprep

Electrophoresis

Gel Extraction

Electrophoresis

Transformation

Ligation

Colony PCR