Template:Kyoto/Notebook/Sep 6

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Sep 6

Electrophoresis

Hirano

Lane	Sample
1	100bp ladder
2	9/4 RBS-lysis3-DT -3
3	9/4 RBS-lysis3-DT -4
4	9/4 RBS-lysis3-DT -5
5	9/4 RBS-lysis1-DT -3
6	9/4 Ptrc-KaiC -1
7	9/4 pSB4K5 -1
8	100bp ladder

File:Igku xxxxxx.xxx

Colony PCR

No name

Sample	base pair
Pconst-spinach-DT-(1~4)	563
Plac-spinach-DT-(1~4)	659
Pcon-pT181 antisense-(1~4)	468
Ptet-pT181 antisense-(1~4)	604
Pconst-pT181 attenuater-(1~4)	568
Pconst+RBS-tetR-DT-(1~4)	1121
Ptet+RBS-lacZα-DT-(1~4)	1162

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	40s	30cycles

Liquid Culture

No name

Sample	medium
9/4 RBS-Lysis3+DT3	Plusgrow medium(+CP)
9/4 RBS-Lysis1+DT3	Plusgrow medium(+CP)
9/4 pSB4K5	Plusgrow medium(+Kan)

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Pconst-spinach-DT -(1)
3	Pconst-spinach-DT -(2)
4	Pconst-spinach-DT -(3)
5	Pconst-spinach-DT -(4)
6	Plac-spinach-DT -(1)
7	Plac-spinach-DT -(2)
8	Plac-spinach-DT -(3)
9	Plac-spinach-DT -(4)
10	Pcon-pT181 antisense -(1)
11	Pcon-pT181 antisense -(2)
12	Pcon-pT181 antisense -(3)
13	Pcon-pT181 antisense -(4)

File:Igku xxxxxx.xxx

Lane	Sample
1	100bp ladder
2	Ptet-pT181 antisense -(1)
3	Ptet-pT181 antisense -(2)
4	Ptet-pT181 antisense -(3)
5	Ptet-pT181 antisense -(4)
6	Plac-pT181 attenuater -(1)
7	Plac-pT181 attenuater -(2)
8	Plac-pT181 attenuater -(3)
9	Plac-pT181 attenuater -(4)

File:Igku xxxxxx.xxx

Miniprep

Honda and Kojima

DNA	concentration [µg/mL]	260/280	260/230
pT181 antisense-(1)	238.7	1.68	1.44
pT181 antisense-(2)	248.8	1.83	1.77
pT181 antisense-(3)	223.2	1.78	1.61
pT181 antisense-(4)	220.8	1.89	1.92
tetR aptamer-(1)	252.5	1.89	1.90
tetR aptamer-(2)	211.2	1.90	1.99
DT181 attenuater	102.2	1.98	2.17
121R aptamer-DT	251.7	1.92	2.01
pSB4K5	212.9	1.80	1.90
RBS-lacZα-DT	185.7	1.78	1.79
RBS-lysis2-DT-(1)	84.8	1.85	2.24

Restriction Enzyme Digestion

Tatsui

	9/6 RBS-lysis2-DT (78.3 µg/mL)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	22 µL	1 µL	1 µL	3 µL	3 µL	0 µL	30 µL
NC	1.3 µL	0 µL	0 µL	1 µL	1 µL	6.7 µL	10 µL

	aptamer-DT (251.7 µg/mL)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	0.4 µL	1 µL	1 µL	3 µL	3 µL	14.1 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	Pλ-luxI (86.4 µg/mL)	EcoRI	SpeI	BSA	Buffer	MilliQ	total
2 cuts	23.1 µL	1 µL	1 µL	3 µL	3 µL	1.9 µL	30 µL
NC	1.2 µL	0 µL	0 µL	1 µL	1 µL	6.8 µL	10 µL

at 37 °C, for 1h

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	RBS-lysis2-DT	XbaI & PstI
3	RBS-lysis2-DT	XbaI & PstI
4	--	--
5	aptamer 12_1R-DT	XbaI & PstI
6	aptamer 12_1R-DT	XbaI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pλ-luxI	EcoRI & SpeI
3	Pλ-luxI	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis2-DT (XbaI & PstI)	7.1	1.88	0.07
aptamer 12_1R-DT (XbaI & PstI)	15.3	1.93	0.70
Pλ-luxI (EcoRI & SpeI)	4.5	1.79	0.31

Liquid Culture

No name

Sample	medium
9/5　Pconst-spinach-DT-(1)	Plusgrow medium(+Amp&CP)
9/5　Ptet-pT181 antisense-(1)	Plusgrow medium(+CP)
9/5　Pconst+RBS-tetR-DT-(1)	Plusgrow medium(+Amp&CP)
9/5　Ptet+RBS-lacZα-DT-(1)	Plusgrow medium(+CP)
9/5　Pcon-pT181 attenuater-(1)	Plusgrow medium(+CP)
9/4　pT181 antisense-(1)	Plusgrow medium(+CP)
9/4　aptamer 12-1R-(1)	Plusgrow medium(+CP)

Colony PCR

No name

Sample	base pair
9/5 Plac-spinach-DT-(5~12)	563
9/5 Pcon-antisense-(5~12)	468
9/5 Plac-attenuater-(5~12)	664
9/5 pT181 antisense+Plac-(1)	-

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

Template:Kyoto/Notebook/Sep 6

From 2013.igem.org

Contents

Sep 6

Electrophoresis

Colony PCR

Liquid Culture

Electrophoresis

Miniprep

Restriction Enzyme Digestion

Gel Extraction

Liquid Culture

Colony PCR