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Aug 21

Plusgrow Medium

Tatsui

material

8/17 Plusgrow medium 50ml
7/22 5000x　Ampicillin 10µl

Measuring materials and putting them in a 50ml tube

Liquid culture

Tatsui

Sample	medium
8/20 tRNA-spinach -(1)	8/21 Plusgrow medium(+Amp)
8/20 tRNA-spinach -(2)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(1)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(2)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(1)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(2)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(1)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(2)	8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(1)	8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(2)	8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(1)	8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(2)	8/21 Plusgrow medium(+Amp)

incubate 37°C 10hour

Gel Extraction

Nakamoto and TaTsui

Lane	DNA	Enzyme
1	1kbp ladder	--
2	8/20 Ptet -(1)	EcoRI & XbaI
3	8/20 Ptet -(1)	EcoRI & XbaI
5	8/20 RBS-tetR-DT -(2)	EcoRI & SpeI
6	8/20 RBS-tetR-DT -(2)	EcoRI & SpeI
8	8/20 Pconst-RBS-GFP-DT -(1)	EcoRI & SpeI
9	8/20 Pconst-RBS-GFP-DT -(1)	EcoRI & SpeI
11	8/20 RBS-lysis3 -(1)	EcoRI & SpeI
12	8/20 RBS-lysis3 -(1)	EcoRI & SpeI

[fig bifore][fig after]

Name	concentration[µg/mL]	260/280	260/230
Ptet (EcoRI & XbaI)	21	1.55	0.78
RBS-tetR-DT (EcoRI & SpeI)	12	1.45	0.62
Pconst-RBS-GFP-DT (EcoRI & SpeI)	11	1.36	0.60
RBS-lysis3 (EcoRI & SpeI)	10	1.26	0.50

Ristriction Enzyme Digestion

Kojima and Nakamoto

8/20 Ptet-(1)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
4.5	1	1	3	3	17.5	30
0.5	0.2	0	1	1	7.3	10
0.5	0	0.2	1	1	7.3	10
0.5	0	0	1	1	7.5	10

8/20 Pconst-RBS-luxR-DT-(2)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2.9	1	1	3	3	19.1	30
0.3	0.2	0	1	1	7.5	10
0.3	0	0.2	1	1	7.5	10
0.3	0	0	1	1	7.7	10

8/17 DT	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
5.3	1	1	3	3	16.7	30
0.5	0.2	0	1	1	7.3	10
0.5	0	0.2	1	1	7.3	10
0.5	0	0	1	1	7.5	10

incubate 37°C 1h

Electrophoresis

Kojima and Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	8/20 Ptet -(1)	EcoRI	XbaI
3	8/20 Ptet -(1)	EcoRI	--
4	8/20 Ptet -(1)	--	XbaI
5	8/20 Ptet -(1)	--	--
6	8/20 J23100-RBS-luxR-DT -(2)	EcoRI	XbaI
7	8/20 J23100-RBS-luxR-DT -(2)	EcoRI	--
8	8/20 J23100-RBS-luxR-DT -(2)	--	XbaI
9	8/20 J23100-RBS-luxR-DT -(2)	--	--
10	8/20 DT	EcoRI	XbaI
11	8/20 DT	EcoRI	--
12	8/20 DT	--	XbaI
13	8/20 DT	--	--
14	1kbp ladder	--	--
15	8/20 Ptet -(1)	EcoRI	XbaI

The reason why lane 2 and 15 were same is that the well of lane 2 might be broken.

[fig]
Kojima

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	8/20 J23100-RBS-luxR-DT -(2)	EcoRI	XbaI
3	8/20 DT	EcoRI	XbaI

[fig]

Gel Extraction

Honda

Lane	DNA	Enzyme
1	1kb ladder	--
2	8/21 J23100-RBS-luxR-DT 25µL	E+X
3	8/21 J23100-RBS-luxR-DT 25µL	E+X
4	--	--
5	8/21 Ptet 25µL	E+X
6	8/21 Ptet 25µL	E+X

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Electrophoresis

Honda

Lane	Sample	EcoRI	XbaI
1	1kb ladder	--	--
2	8/21 DT	+	+
3	8/21 DT	+	--
4	8/21 DT	--	+
5	8/21 DT	--	--

File:Igku xxxxxx.xxx

Ligation

No name

Name	-?-	NC
8/21 Pcn-RBS-luxR-DT(+Amp)	2.3	2.3
8/21 Pcn-RBS-GFP-DT	1.7	0
Ligation High buffer	2	2
MilliQ	0	1.7
total	6	6

Name	-?-	NC
8/18 Plux(+CP)	1.1	1.1
8/19 RBS-GFP-DT	5	0
Ligation High buffer	3.1	3.1
MilliQ	0	5
total	9.2	9.2

Name	-?-	NC
8/18 RBS(+Amp)	2.2	--
8/19 lysis1	6.0	--
Ligation High buffer	4.1	--
MilliQ	0	--
total	12.3	--

Name	-?-	NC
8/18 RBS(+Amp)	2.2	2.2
8/19 lysis2	3.6	0
Ligation High buffer	2.9	2.9
MilliQ	0	3.6
total	8.7	8.7

Transformation

Okazaki

Name	Sample	Competent Cells	Total	Plate
8/21 Pcon-RBS-GFP-DT+Pcon-RBS-GFP-DT	1µL	10µL	11µL	Amp
8/21 Pcon-RBS-GFP-DT NC	1µL	10µL	11µL	Amp
8/18 RBS+8/19 lysis1	1µL	10µL	11µL	Amp
8/18 RBS+8/19 lysis2	1µL	10µL	11µL	Amp
8/18 RBS NC	1µL	10µL	11µL	Amp
8/18 Plux+8/18 RBS-GFP-DT	1µL	10µL	11µL	CP
8/18 Plux+8/18 RBS-GFP-DT	1µL	10µL	11µL	CP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2)	1µL	10µL	11µL	CP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2) NC	1µL	10µL	11µL	CP
8/21 pSB1C3	1µL	10µL	11µL	CP

LB Medium Plate

Gel Extraction

Okazaki

Lane	DNA	Enzyme
1	1kb ladder	3µL
2	--	--
3	8/21 DT E+X	12.5µL
4	8/21 DT E+X	12.5µL

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Miniprep

Tatsui, Kojima, and Nakamoto

DNA	concentration [µg/mL]	260/280	260/230
tRNA-spinach-(1)	234	1.64	2.14
tRNA-spinach-(2)	440	1.60	1.91
tetR-aptamer 12-p(1076)-(1)	150	1.61	2.03
tetR-aptamer 12-p(1076)-(2)	374	1.45	1.66
tetR-aptamer 12-1R(113)-(1)	304	1.68	2.21
tetR-aptamer 12-1R(113)-(2)	382	1.47	1.66
tetR-aptamer 12-1M(105)-(1)	374	1.68	2.20
tetR-aptamer 12-1M(105)-(2)	286	1.31	1.43
PT181 attenuator-(1)	330	1.66	2.11
PT181 attenuator-(2)	270	1.68	2.18
Fusion1 attenuator-(1)	306	1.67	2.20
Fusion1 attenuator-(2)	282	1.60	1.94
Fusion3m2 attenuator-(1)	332	1.68	2.25
Fusion3m2 attenuator-(2)	326	1.67	2.10
PT181 antisense-(1)	178	1.64	1.68
PT181 antisense-(2)	116	1.63	1.95
Fusion1 antisense-(1)	300	1.67	2.21
Fusion1 antisense-(2)	164	1.42	1.46
Fusion6 antisense-(1)	336	1.65	2.18
Fusion6 antisense-(2)	192	1.65	2.01

Template:Kyoto/Notebook/Aug 21

From 2013.igem.org

Contents

Aug 21

Plusgrow Medium

Liquid culture

Gel Extraction

Ristriction Enzyme Digestion

Electrophoresis

Gel Extraction

Electrophoresis

Ligation

Transformation

LB Medium Plate

Gel Extraction

Miniprep