Team:Kyoto/Notebook

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template

Transformation

じっけんしゃ

Name	Well	Sample	Competent Cells	Total	Plate
いでんし	ぱーつのうぇる	なんちゃらµL	ごにょごにょµL	ほにゃららµL	こーせーぶっしつ
いかどうよう

Restriction Enzyme Digestion

じっけんしゃ

DNA	Buffer	restricion enzyme	restriction enzyme	MilliQ	total
（°°） µL	µL	µL	µL	µL	µL

Electrophoresis

じっけんしゃ

Lane	Sample	Enzyme1	Enzyme2
（°Д°）

File:Igku xxxxxx.xxx

Ligation

じっけんしゃ

Name	目的（番号？）？	NCとか µL
(＞o＜)

Gel Extraction

じっけんしゃ

Lane	DNA	Enzyme
（＞ω・）b

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

Name	quantity	concentration[µg/mL]	260/280	260/230
（´ω｀）
（ΦωΦ^）

Colony PCR

No name

Sample	base pair
(｀・ω・´)

PreDenature	Denature	Annealing	Extension	cycle
°C	°C	°C	°C	--
（◎Д◎）

Liquid Culture

No name

Sample	medium
(・ω・)
（・Ｘ・）

Miniprep

DNA	concentration[µg/mL]	260/280	260/230
(・▽・)

Master Plate

No name

Number	Use LB plate (+抗生物質)
ʅ（´◔౪◔）ʃ

Aug 19

Colony PCR

Kojima

Sample	base pair
8/18 J23100 control -(1)	1142
8/18 J23100-RBS-GFP-DT -(1)	1156
8/18 J23100-RBS-GFP-DT -(2)	1156
8/18 J23100-RBS-luxR-DT -(1)	1271
8/18 J23100-RBS-luxR-DT -(2)	1271
8/18 J23100-RBS-lacZα-DT -(1)	670
8/18 J23100-RBS-lacZα-DT -(2)	670
8/18 RBS-lysis3 -(1)	997
8/18 RBS-lysis3 -(2)	997
8/18 P0440(RBS-tetR-DT) -(1)	1154
8/18 P0440(RBS-tetR-DT) -(2)	1154
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

File:Igku Aug19electrophoresis1.png

Sample	base pair
8/18 RBS control -(1)	--
8/18 R0040(Ptet) -(1)	686
8/18 R0040(Ptet) -(2)	686
negative control	--

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

File:Igku Aug19electrophoresis2.png

Gel Extraction

Ashida

Lane	DNA	Enzyme
1	100bp ladder	--
3	8/18 K117000(lysis2) -1	XbaI & PstI
5	8/18 K117000(lysis2) -1	XbaI & PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
8/18 lysis2-1(XbaI & PstI)	3.1	3.64	0.19
8/18 lysis2-1(XbaI & PstI)	3.4	1.89	0.13

LB Medium Plate

Nakamoto

volume	200ml
Bacto T2ypton	2g
Bacto yeast extract	1g
NaCl	1g
Agar Pouder	2g

Restriction Enzyme Digestion

Tatsui

lysis2-1	XbaI	PstI	BSA	10xbuffer	MilliQ	total
10µL	1µL	1µL	3µL	3µL	12µL	30µL
2µL	0.2µL	--	1µL	1µL	5.8µL	10µL
2µL	--	0.2µL	1µL	1µL	5.8µL	10µL
2µLL	--	--	1µL	1µ	6µL	10µL

at 37°C, for 1h

Murata and Okazaki

8/19 lysis1-1	10xbuffer	XbaI	PstI	BSA	MilliQ	total
2µL	2µL	0.5µL	0.8µL	2µL	13µL	20µL
0.5µL	1µL	0.2µL	--	1µL	7.3µL	10µL
0.5µL	1µL	--	0.2µL	1µL	7.3µL	10µL
0.5µL	1µL	--	--	1µL	7.5µL	10µL

at 37°C, for 1h

Ligation

No name

Name		NC
RBS	0.3	0.3
lysis2	7.4	--
MilliQ	--	7.4

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/19 lysis1	XbaI	PstI
3	8/19 lysis1	XbaI	--
4	8/19 lysis1	--	PstI
5	8/19 lysis1	--	--
6	8/19 lysis1(Gel Extraction Product)	XbaI	PstI

Transformation

Murata and Kojima

Name	Well	Sample	Competent Cells	Plate
8/18 Plux+RBS-GFP-DT	--	2µL	20µL	CP
8/18 Plux(NC)	--	2µL	20µL	CP
8/19 RBS+lysis2	--	2µL	20µL	Amp
8/15 Pbad-araC	Plate5-14N	2µL	20µL	Kan

Incubate at 37°C

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	8/19 lysis1 ①	XbaI & PstI

[fig before] [fig after]

Name	concentration[µg/mL]	260/280	260/230
8/19 lysis1 ①(XbaI & PstI)	87.8	1.06	0.82

Liquid Culture

Ashida

Sample	medium
8/18 Ptet -1	Plusgrow medium(+CP)
8/18 Ptet -2	Plusgrow medium(+CP)
8/18 RBS-tetR-DT -1	Plusgrow medium(+CP)
8/18 RBS-tetR-DT -2	Plusgrow medium(+CP)
8/18 J23100-GFP -1	Plusgrow medium(+Amp)
8/18 J23100-luxR -1	Plusgrow medium(+Amp)
8/18 J23100-luxR -2	Plusgrow medium(+Amp)
8/18 J23100-lacZα -1	Plusgrow medium(+Amp)
8/18 RBS-lysis3 -1	Plusgrow medium(+Amp)
8/18 RBS-lysis3 -2	Plusgrow medium(+Amp)

Master Plate

Ashida

Number	Use LB plate(+Amp)
1	8/18 J23100-GFP -1
2	8/18 J23100-luxR -1
3	8/18 J23100-luxR -2
4	8/18 J23100-lacZα -1
5	8/18 RBS-lysis3 -1
6	8/18 RBS-lysis3 -2

Number	Use LB plate(+CP)
1	8/18 Ptet -1
1	8/18 Ptet -2
3	8/18 RBS-tetR-DT -1
4	8/18 RBS-tetR-DT -2

Aug 20

Miniprep

Kojima

DNA	concentration[µg/µL]	260/280	260/230
8/18 Ptet -1	220	1.69	1.87
8/18 Ptet -2	210	1.69	1.71
8/18 RBS-tetR-DT -1	236	1.63	1.69
8/18 RBS-tetR-DT -2	182	1.65	1.98
8/18 J23100-RBS-GFP-DT -1	334	1.71	2.12
8/18 J23100-RBS-luxR-DT -1	440	1.67	1.60
8/18 J23100-RBS-luxR-DT -2	344	1.68	1.83
8/18 J23100-RBS-lacZα-DT -1	280	1.70	1.81
8/18 RBS-lysis3 -1	282	1.67	1.76
8/18 RBS-lysis3 -2	212	1.30	1.35

LB Medium Plate

Okazaki

volume	200mL
Bacto Trypton	2g
Bacto yeast extract	1g
Nacl	1g
Agar Powder	2g
Amp	40µl

1. Measuring reagents and put they in the Erlenmeyer flask.
2. diluting in measuring cylinder to 200ml total.
3. autoclaving.
4. adding ampicirine 40ml.

Ristriction Enzyme Digestion

Kojima and Ashida

8/20 Ptet -1(220µg/mL)	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 RBS-tetR-DT -2(182µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
5.5	1	1	3	19.5	30
0.6	0.2	0	1	8.2	10
0.6	0	0.2	1	8.2	10
0.6	0	0	1	8.4	10

8/20 J23100-RBS-GFP-DT(334µg/ml)	EcoRI	SpeI	10x buffer H	MilliQ	total
3.0	1	1	3	22	30
0.3	0.2	0	1	8.5	10
0.3	0	0.2	1	8.5	10
0.3	0	0	1	8.7	10

8/20 J23100-RBS-luxR-DT -2(344µg/mL)	EcoRI	XbaI	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

8/20 RBS-lysis3 -1(282µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
3.5	1	1	3	21.5	30
0.4	0.2	0	1	8.4	10
0.4	0	0.2	1	8.4	10
0.4	0	0	1	8.6	10

8/17 DT -1(188µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
5.3	1	1	3	3	16.7	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

Transformation

Nakamoto

Name	Sample	Competent Cells(XL10-gold)	Total	Plate
tRNA-Spinach-tRNA	1µL	10µL	11µL	Amp
tetR-aptamer 12_PC(076)	1µL	10µL	11µL	Amp
tetR-aptamer 12_1R(113)	1µL	10µL	11µL	Amp
tetR-aptamer 12_1M(105)	1µL	10µL	11µL	Amp
pT181 attenuator	1µL	10µL	11µL	Amp
Fusin1 attenuator	1µL	10µL	11µL	Amp
Fusion3m2 attenuator	1µL	10µL	11µL	Amp
pT181 antisense	1µL	10µL	11µL	Amp
Fusion1 antisense	1µL	10µL	11µL	Amp
Fusion6 antisense	1µL	10µL	11µL	Amp

incubate 37°C overnight 20:34~

Electrophoresis

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	8/20 Ptet -1	EcoRI	XbaI
2	8/20 Ptet -1	EcoRI	--
3	8/20 Ptet -1	--	XbaI
4	8/20 Ptet -1	--	--
5	8/20 RBS-tetR-DT -2	EcoRI	SpeI
6	8/20 RBS-tetR-DT -2	EcoRI	--
7	8/20 RBS-tetR-DT -2	--	SpeI
8	8/20 RBS-tetR-DT -2	--	--
9	1kbp ladder	--	--
10	8/20 J23100-RBS-GFP-DT -1	EcoRI	SpeI
11	8/20 J23100-RBS-GFP-DT -1	EcoRI	--
12	8/20 J23100-RBS-GFP-DT -1	--	SpeI
13	8/20 J23100-RBS-GFP-DT -1	--	--
14	8/20 J23100-RBS-luxR-DT -1	EcoRI	XbaI
15	8/20 J23100-RBS-luxR-DT -1	EcoRI	--
16	8/20 J23100-RBS-luxR-DT -1	--	XbaI
17	8/20 J23100-RBS-luxR-DT -1	--	--
18	1kbp ladder	--	--
19	8/20 RBS-lysis3 -1	EcoRI	SpeI
20	8/20 RBS-lysis3 -1	EcoRI	--
21	8/20 RBS-lysis3 -1	--	SpeI
22	8/20 RBS-lysis3 -1	--	--
23	1kbp ladder	--	--
24	8/17 DT	EcoRI	XbaI
25	8/17 DT	EcoRI	--
26	8/17 DT	--	XbaI
27	8/17 DT	--	--

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

Aug 22

Liquid culture

Nakamoto

Sample	Medium
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1)	8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2)	8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (1)	8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (2)	8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (1)	8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (2)	8/21 Plusgrow medium (+Amp)
8/21 RBS control (1)	8/21 Plusgrow medium (+Amp)
8/21 RBS control (2)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (1)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (2)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (1)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (2)	8/21 Plusgrow medium (+Amp)
8/21 Plux-RBS-GFP-DT (1)	8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (1)	8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (2)	8/21 Plusgrow medium (+Amp)

incubate 37°C 10hour

Colony PCR

Kojima

Sample	base pair
8/21 RBS-lysis1(1)	400
8/21 RBS-lysis1(2)	400
8/21 RBS control(1)	--
8/21 RBS control(2)	--
negative control	--

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

Sample	base pair
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1)	2143
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2)	2143
8/21 Pcon-RBS-luxR control(1)	--
8/21 Pcon-RBS-luxR control(2)	--
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycles

Sample	base pair
8/21 Ptet-RBS-tetR-DT (1)	1216
8/21 Ptet-RBS-tetR-DT (2)	1216
8/21 Ptet control (1)	--
8/21 Ptet control (2)	--
8/21 Plux-RBS-GFP-DT (1)	1227
8/21 pSB1C3(BBa_J04450) (1)	1353
8/21 pSB1C3(BBa_J04450) (2)	1353
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	pT181antisense	EcoRI	SpeI
2	pT181antisense	EcoRI	--
3	pT181antisense	--	SpeI
4	pT181antisense	--	--
5	pT181antisense	XbaI	PstI
6	pT181antisense	XbaI	--
7	pT181antisense	--	PstI
8	pT181antisense	--	--
9	100bp ladder	--	--
10	Fusion1 attenuator	EcoRI	SpeI
11	Fusion1 attenuator	EcoRI	--
12	Fusion1 attenuator	--	SpeI
13	Fusion1 attenuator	--	--
14	Fusion1 attenuator	XbaI	PstI
15	Fusion1 attenuator	XbaI	--
16	fusion1 attenuator	--	PstI
17	fusion1 attenuator	--	--

File:Igku xxxxxx.xxx

Lane	Sample	Enzyme1	Enzyme2
1	Fusion3 3m2 attenuator	EcoRI	SpeI
2	Fusion3 3m2 attenuator	EcoRI	--
3	Fusion3 3m2 attenuator	--	SpeI
4	Fusion3 3m2 attenuator	--	--
5	Fusion3 3m2 attenuator	XbaI	PstI
6	Fusion3 3m2 attenuator	XbaI	--
7	Fusion3 3m2 attenuator	--	PstI
8	Fusion3 3m2 attenuator	--	--
9	100bp ladder	--	--
10	aptamer 12-1M	EcoRI	SpeI
11	aptamer 12-1M	EcoRI	--
12	aptamer 12-1M	--	SpeI
13	aptamer 12-1M	--	--
14	aptamer 12-P	EcoRI	SpeI
15	aptamer 12-P	EcoRI	--
16	aptamer 12-1P	--	SpeI
17	aptamer 12-1P	--	--

Team:Kyoto/Notebook

From 2013.igem.org

Contents

template

Transformation

Restriction Enzyme Digestion

Electrophoresis

Ligation

Gel Extraction

Colony PCR

Liquid Culture

Miniprep

Master Plate

Aug 19

Colony PCR

Gel Extraction

LB Medium Plate

Restriction Enzyme Digestion

Ligation

Electrophoresis

Transformation

Gel Extraction

Liquid Culture

Master Plate

Aug 20

Miniprep

LB Medium Plate

Ristriction Enzyme Digestion

Transformation

Electrophoresis

Aug 21

Plusgrow Medium

Liquid culture

Gel Extraction

Ristriction Enzyme Digestion

Electrophoresis

Gel Extraction

Electrophoresis

Ligation

Transformation

LB Medium Plate

Gel Extraction

Miniprep

Aug 22

Liquid culture

Colony PCR

Electrophoresis

Restriction Enzyme Digestion

Miniprep

Electrophoresis

Aug 23

Electrophoresis

Restriction Enzyme Digestion

Electrophoresis