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Calendar

July
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13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

August
					1	2
3	4	5	6	7	8	9
10	11	12	13	14	15	16
17	18	19	20	21	22	23
24	25	26	27	28	29	30
31

September
	1	2	3	4	5	6
7	8	9	10	11	12	13
14	15	16	17	18	19	20
21	22	23	24	25	26	27
28	29	30

Octorber
			1	2	3	4
5	6	7	8	9	10	11
12	13	14	15	16	17	18
19	20	21	22	23	24	25
26	27	28	29	30	31

アメフラシはこちら

7/15

transformation

Tatsui, Matsumoto and Sukegawa

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J23101(Pcon)	2	20(2/23)	22	LB(CP)
BBa_B0032(RBS)	2	20(2/23)	22	LB(CP)

7/16

transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_B0030(1)	2	20(2/23)	22	LB(CP)
BBa_B0030(2)	2	20(bought)	22	LB(CP)
BBa_B0032(1)	2	20(2/23)	22	LB(CP)
BBa_B0032(2)	2	20(bought)	22	LB(CP)

7/17

Colony PCR

Tatsui, Matsumoto, Li, and Yamauchi

NameⅠ	NameⅡ
7/16 BBa_B0030(1) Colony_1	7/16 BBa_B0032(1) Colony_1
7/16 BBa_B0030(1) Colony_2	7/16 BBa_B0032(1) Colony_2
7/16 BBa_B0030(1) Colony_3	7/16 BBa_B0032(1) Colony_3
7/16 BBa_B0030(2) Colony_1	7/16 BBa_B0032(2) Colony_1
7/16 BBa_B0030(2) Colony_2	7/16 BBa_B0032(2) Colony_2
7/16 BBa_B0030(2) Colony_3	7/16 BBa_B0032(2) Colony_3
Negative Control	-
-	-

Liquid Culture

Tatsui, Matsumoto, Li, and Yamauchi

Name	medium
7/16 BBa_B0030(1) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0030(1) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0030(1) Colony_3	Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_3	Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_3	Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_1	Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_2	Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_3	Plusgrow medium(CP)

7/18

Miniprep

Tatsui, Matsumoto, Tsujii, Stephane, and Yamauchi

DNA	Concentration/(µg/ml)	260/280	260/230
7/16 BBa_B0030(1) Colony_1	229	1.96	1.40
7/16 BBa_B0030(1) Colony_2	209	1.95	1.50
7/16 BBa_B0030(1) Colony_3	108	2.12	2.12
7/16 BBa_B0030(2) Colony_1	-	-	-
7/16 BBa_B0030(2) Colony_2	174	2.02	1.56
7/16 BBa_B0030(2) Colony_3	158	2.01	1.68
7/16 BBa_B0032(1) Colony_1	179	2.00	2.06
7/16 BBa_B0032(1) Colony_2	-	-	-
7/16 BBa_B0032(1) Colony_3	221	1.90	1.37
7/16 BBa_B0032(2) Colony_1	225	1.89	1.37
7/16 BBa_B0032(2) Colony_2	270	1.98	1.63
7/16 BBa_B0032(2) Colony_3	194	1.89	1.33

7/30

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	EcoR1/(µl)	Xba1/(µl)	Spe1/(µl)	Pst1/(µl)	Buffur/(µl)	BSA/(µl)	MilliQ/(µl)	TOTAL/(µl)
7/16 BBa_B0030(2) Colony_1	1	0.3	0	0	0	1(H)	0	7.7	10
7/16 BBa_B0030(2) Colony_1	1	0	0.3	0	0	1(M)	1	6.7	10
7/16 BBa_B0030(2) Colony_1	1	0	0	0.3	0	1(M)	0	7.7	10
7/16 BBa_B0030(2) Colony_1	1	0	0	0	0.3	1(H)	0	7.7	10
7/16 BBa_B0030(2) Colony_1(Nega)	1	0	0	0	0	1(M)	0	8	10

Electrophoresis

No name

Lane	Restriction Enzyme Digestion Products
1	7/30 BBa_B0030(2) Colony_1(E CUT)
2	7/30 BBa_B0030(2) Colony_1(X CUT)
3	7/30 BBa_B0030(2) Colony_1(S CUT)
4	7/30 BBa_B0030(2) Colony_1(P CUT)
5	7/30 BBa_B0030(2) Colony_1(Nega)
6	1kbp ladder

7/31

Transformation

Hashitani, Tatsui, Li and Yamauchi

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_E0240(RBS-GFP-DT)	2	20	22	LB(CP)
J23100(Pcon)	2	20	22	LB(AMP)

Liquid Culture

No name

Name	medium
7/31 J23100(Pcon) Colony_1	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_2	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_3	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_4	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_5	Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_6	Plusgrow medium(AMP)

Restriction Enzyme Digestion

Hashitani, Tatsui, Li and Yamauchi

7/18 RBS Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Pst1/(µl)	Xba1/(µl)	Buffur/(µl)	BSA/(µl)	MilliQ/(µl)	TOTAL/(µl)
BBa_B0032	5	0.3	0	0	0	1(H)	0	3.7	10
BBa_B0032	5	0	0.3	0	0	1(M)	0	3.7	10
BBa_B0032	5	0	0	0.3	0	1(H)	0	3.7	10
BBa_B0032	5	0	0	0	0.3	1(M)	1	2.7	10
BBa_B0032(Nega)	5	0	0	0	0	1(M)	0	4	10

8/1

Electrophoresis

Honda, Li and Yamauchi

Lane	Restriction Enzyme Digestion Products
1	1kb ladder
2	7/31 BBa_B0032(E CUT)
3	7/31 BBa_B0032(S CUT)Error
4	-
5	-
6	7/31 BBa_B0032(P CUT)
7	7/31 BBa_B0032(X CUT)
8	7/31 BBa_B0032(Nega)

PCR

Tatsui

BBa_E0240(RBS-GFP-DT)	Volume
10×buffur/(µl)	5
2mMdNTPs/(µl)	5
25mM MgSO4/(µl)	2
VF Primer/(µl)	1.5
VR Primer/(µl)	1.5
template/(µl)	0.5
KOD-plus/(µl)	1
MilliQ/(µl)	33.5
TOTAL/(µl)	50

Steps	Temparature	Time	cycle
Pre Denature	94℃	2min	1time
Denature	94℃	15sec	30cycle
Annealing	55℃	30sec	30cycle
Extension	68℃	1min	30cycle

DNA	Concentration/(µg/ml)	260/280	260/230
Product	80	1.91	1.78

8/2

Miniprep

Tatsui, Honda, Matsumoto, Yamauchi, Li, Tsujii, and Nakamura

DNA	Concentration/(µg/ml)	260/280	260/230
7/31 J23100(Pcon) Colony_1	145	2.11	3.09
7/31 J23100(Pcon) Colony_2	-	-	-
7/31 J23100(Pcon) Colony_3	160	2.01	3.13
7/31 J23100(Pcon) Colony_4	140	1.87	2.70
7/31 J23100(Pcon) Colony_5	105	2.39	5.90
7/31 J23100(Pcon) Colony_6	180	1.90	1.37

8/20

Transformation

Honda

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
7/31 BBa_E0240(RBS-GFD-DT)	2	20	22	LB(CP)
BBa_I732095	2	20	22	LB(CP)

8/21

Colony PCR

Shimazaki

NameⅠ
8/20 BBa_I732095 Colony_1
8/20 BBa_I732095 Colony_2
8/20 BBa_I732095 Colony_3
Negative control

Electrophoresis

Honda

Lane	PCR Product
1	1kb ladder
2	8/21 BBa_I732095 Colony_1
3	8/21 BBa_I732095 Colony_2
4	8/21 BBa_I732095 Colony_3
5	Negative Control

Liquid Culture

Honda and Tatsui

Name	medium
8/20 BBa_I732095 Colony_1	Plusgrow medium(CP)
8/20 BBa_I732095 Colony_2	Plusgrow medium(CP)
8/20 BBa_I732095 Colony_3	Plusgrow medium(CP)

8/22

Miniprep

Honda

DNA	Concentration/(µg/ml)	260/280	260/230
8/20 BBa_I732095 Colony_1	430	1.68	2.13
8/20 BBa_I732095 Colony_2	255	1.87	1.81
8/20 BBa_I732095 Colony_3	215	1.78	1.35

Restriction Enzyme Digestion

Nakamura

Sample	DNA/(µl)	EcoR1/(µl)	Spe1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
8/22 BBa_I732095 Colony_1	4.7	1	1	3(H)	20.3	30
8/22 BBa_I732095 Colony_2	7.8	1	1	3(H)	17.2	30
8/22 BBa_I732095 Colony_3	9.3	1	1	3(H)	15.7	30

Transformation

Tatsui and Yamauchi

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB3C5)	2	20	22	LB(CP)

8/25

Transformation

Tatsui, Kita and Li

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB2K3)	2	20	22	LB(KAN)
BBa_C0051(pSB1A2)	2	20	22	LB(AMP)

Liquid Culture

Tatsui

Name	medium
8/22 BBa_J04450(pSB3C5)	Plusgrow medium(CP)*5

8/26

Miniprep

Tsujii, Honda and Matsumoto

DNA	Concentration/(µg/ml)	260/280	260/230
8/25 BBa_J04450(pSB3C5)	575	1.37	1.37

Liquid Culture

Tatsui, Matsumoto and Nakamura

Name	medium
8/25 BBa_J04450(pSB2K3)	Plusgrow medium(KAN)*5
8/25 BBa_J0051(pSB1A2)	Plusgrow medium(AMP)*2

8/27

Miniprep

No Name

DNA	Concentration/(µg/ml)	260/280	260/230
8/26 BBa_J04450(pSB2K3)	1045	1.43	1.11
8/26 BBa_J0051(pSB1A2)	790	1.39	0.90

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB3T5)	2	20	22	LB(TET)

8/28

Liquid Culture

Yamauchi

Name	medium
8/27 BBa_J04450(pSB3T5)	Plusgrow medium(TET)

8/29

Miniprep

Matsumoto

DNA	Concentration/(µg/ml)	260/280	260/230
8/28 BBa_J04450(pSB3T5)	180	1.60	1.67

Transformation

Tatsui

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
BBa_J04450(pSB4A5)	2	20	22	LB(AMP)

9/1

Colony PCR

NameⅠ
-
-
-
9/1 pSB4A5 Colony_1
9/1 pSB4A5 Colony_2
Negative Control

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
8/28 Miniprep pSB3T5	25	2	2	6(H)	25	60
Negative control_1	3.7	0	0	1(H)	5.3	10
8/22 Miniprep I732095	14	3	3	9(H)	61	90
Negative control_2	1.5	0	0	1(H)	7.5	10
8/27 Miniprep pSB2K3	6	3	3	9(H)	69	90
Negative control_3	1	0	0	1(H)	8	10
8/2 Miniprep Pcon Colony_3	20	1.5	1.5	5(H)	17	45
Negative control_4	4	0	0	1(H)	5	10
8/2 Miniprep Pcon Colony_1	20	1.5	1.5	5(H)	17	45
Negative control_5	4	0	0	1(H)	5	10

Liquid Culture

No name

Name	medium
8/29 BBa_J04450(pSB4A5) Colony_1	Plusgrow medium(AMP)*2
8/1 Pcon Colony_1	Plusgrow medium(AMP)*2
8/27 pSB3T5 Colony_1	Plusgrow medium(TET)*2

9/2

Miniprep

Honda, Yamauchi and Li

DNA	Concentration/(µg/ml)	260/280	260/230
8/29 BBa_J04450(pSB4A5) Colony_1	210	1.61	1.18
8/1 Pcon Colony_1	215	1.79	1.87
8/27 pSB3T5 Colony_1	215	1.58	0.92

Gel Extractions

Matsumoto

Lane	Sample	Volume/(µl)
1	9/1 pSB3T5 Colony_1 (Nega)	10
2	9/1 pSB3T5 Colony_1 (E+P)(Confirmation)	5
3	9/1 pSB3T5 Colony_1 (E+P)(Extraction)	55
4	1kb ladder	3
5	9/1 I732095 (Nega)	10
6	9/1 I732095 (E+P)(Confirmation)	5
7	9/1 I732095 (E+P)(Extraction)	85
8	1kb ladder	3
9	9/1 pSB2K3 (Nega)	10
10	9/1 pSB2K3 (E+P)(Confirmation)	5
11	9/1 pSB2K3 (E+P)(Extraction)	85

Lane	Sample	Volume/(µl)
1	9/1 Pcon Colony_3 (E+P)(Confirmation)	5
2	9/1 Pcon Colony_3 (E+P)(Extraction)	20
3	9/1 Pcon Colony_3 (E+P)(Extraction)	20
4	9/1 Pcon Colony_3 (Nega)	10
5	1kb ladder	3
6	9/1 Pcon Colony_1 (E+P)(Confirmation)	5
7	9/1 Pcon Colony_1 (E+P)(Extraction)	20
8	9/1 Pcon Colony_1 (E+P)(Extraction)	20
9	9/1 Pcon Colony_1 (Nega)	10

9/3

Concentration Measurement

Matsumoto, Yamauchi and Li

DNA	Concentration/(µg/ml)	260/280	260/230
pSB2K3	40	1.48	0.10
Pcon Colony_1	30	1.76	0.62
pSB3T5	50	2.35	0.50
pSB1C3	65	2.14	0.10
Pcon Colony_3	45	1.56	0.58

Ligation

No name

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
Pcon	2.1	pSB1C3	1.5	1	5	0.4	10
-(Negative Control)	0	pSB1C3	1.5	1	5	2.5	10
Pcon	1.3	pSB2K3	2.5	1	5	0.2	10
-(Negative Control)	0	pSB2K3	2.5	1	5	1.5	10
Pcon	10	pSB3T5	2.3	1	5	0.7	10
-(Negative Control)	0	pSB3T5	2.3	1	5	1.7	10

Transformation

Matsumoto

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/3 Ligation Pcon+pSB1C3	3	15	18	LB(CP)
9/3 Ligation pSB1C3(Negative Control)	8	10	18	LB(CP)
9/3 Ligation Pcon+pSB2K3	3	15	18	LB(KAN)
9/3 Ligation pSB2K3(Negative Control)	8	10	18	LB(KAN)
9/3 Ligation Pcon+pSB3T5	3	15	18	LB(TET)
9/3 Ligation pSB3T5(Negative Control)	2	10	12	LB(TET)

9/4

Colony PCR

Honda and Yamauchi

NameⅠ	NameⅡ	NameⅢ	NameⅣ
-	9/3 pSB1C3(Nega) Colony_7	9/3 Pcon+pSB3T5 Colony_5	9/3 Pcon+pSB2K3 Colony_1
-	9/3 pSB1C3(Nega) Colony_8	9/3 Pcon+pSB3T5 Colony_6	9/3 Pcon+pSB2K3 Colony_2
9/3 pSB1C3(Nega) Colony_1	9/3 Pcon+pSB3T5 Colony_1	9/3 Pcon+pSB3T5 Colony_7	9/3 Pcon+pSB2K3 Colony_3
9/3 pSB1C3(Nega) Colony_2	9/3 Pcon+pSB3T5 Colony_2	9/3 Pcon+pSB3T5 Colony_8	9/3 Pcon+pSB2K3 Colony_4
9/3 pSB1C3(Nega) Colony_3	9/3 Pcon+pSB3T5 Colony_3	9/3 Pcon+pSB3T5 Colony_9	9/3 Pcon+pSB2K3 Colony_5
9/3 pSB1C3(Nega) Colony_4	9/3 Pcon+pSB3T5 Colony_4	9/3 Pcon+pSB3T5 Colony_10	9/3 Pcon+pSB2K3 Colony_6
9/3 pSB1C3(Nega) Colony_5	9/3 pSB1C3(Nega) Colony_9	9/3 pSB3T5(Nega) Colony_1	9/3 Pcon+pSB2K3 Colony_7
9/3 pSB1C3(Nega) Colony_6	9/3 pSB1C3(Nega) Colony_10	9/3 pSB3T5(Nega) Colony_2	9/3 Pcon+pSB2K3 Colony_8

NameⅤ	NameⅥ	NameⅦ	NameⅧ
9/3 Pcon+pSB2K3 Colony_9	9/3 Pcon+pSB1C3 Colony_2	9/3 Pcon+pSB1C3 Colony_10	9/3 pSB1C3(Nega) Colony_8
9/3 Pcon+pSB2K3 Colony_10	9/3 Pcon+pSB1C3 Colony_3	9/3 pSB1C3(Nega) Colony_1	9/3 pSB1C3(Nega) Colony_9
9/3 pSB2K3(Nega) Colony_1	9/3 Pcon+pSB1C3 Colony_4	9/3 pSB1C3(Nega) Colony_2	9/3 pSB1C3(Nega) Colony_10
9/3 pSB2K3(Nega) Colony_2	9/3 Pcon+pSB1C3 Colony_5	9/3 pSB1C3(Nega) Colony_3	9/3 pSB1C3(Nega) Colony_11
9/3 pSB2K3(Nega) Colony_3	9/3 Pcon+pSB1C3 Colony_6	9/3 pSB1C3(Nega) Colony_4	-
9/3 pSB2K3(Nega) Colony_4	9/3 Pcon+pSB1C3 Colony_7	9/3 pSB1C3(Nega) Colony_5	-
9/3 pSB2K3(Nega) Colony_5	9/3 Pcon+pSB1C3 Colony_8	9/3 pSB1C3(Nega) Colony_6	-
9/3 Pcon+pSB1C3 Colony_1	9/3 Pcon+pSB1C3 Colony_9	9/3 pSB1C3(Nega) Colony_7	-

NameⅨ	NameⅩ
9/3 Pcon+pSB1C3 Colony_11	8/22 Miniprep I732095 Colony_3
Negative Control	-

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	EcoR1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
8/27 Miniprep pSB2K3	2	1	1	3(H)	23	30
8/27 Miniprep pSB2K3(Nega)	1	0	0	1(H)	8	10
9/2 Miniprep pSB3T5	10	1	1	3(H)	15	30
9/2 Miniprep pSB3T5(Nega)	3	0	0	1(H)	6	10
9/2 Miniprep pSB4A5	10	1	1	3(H)	15	30
9/2 Miniprep pSB4A5(Nega)	3	0	0	1(H)	6	10
8/22 Miniprep pSB1C3 Colony_2	2	1	1	1(H)	5	10

9/5

Electrophoresis

Honda and Matsumoto

Lane	Colony PCR ProductsⅠ	Lane	Colony PCR ProductsⅠ
1	1kb ladder	10	9/4 pSB1C3(Nega) Colony_7
2	-	11	9/4 pSB1C3(Nega) Colony_8
3	-	12	9/4 pSB1C3(Nega) Colony_9
4	9/4 pSB1C3(Nega) Colony_1	13	9/4 pSB1C3(Nega) Colony_10
5	9/4 pSB1C3(Nega) Colony_2	14	9/4 Pcon+pSB2K3 Colony_1
6	9/4 pSB1C3(Nega) Colony_3	15	9/4 Pcon+pSB2K3 Colony_2
7	9/4 pSB1C3(Nega) Colony_4	16	9/4 Pcon+pSB2K3 Colony_3
8	9/4 pSB1C3(Nega) Colony_5	17	1kb ladder
9	9/4 pSB1C3(Nega) Colony_6

Lane	Colony PCR ProductsⅡ	Lane	Colony PCR ProductsⅡ
1	1kb ladder	10	9/4 pSB3T5(Nega) Colony_2
2	9/4 Pcon+pSB2K3 Colony_4	11	9/4 Pcon+pSB2K3 Colony_1
3	9/4 Pcon+pSB2K3 Colony_5	12	9/4 Pcon+pSB2K3 Colony_2
4	9/4 Pcon+pSB2K3 Colony_6	13	9/4 Pcon+pSB2K3 Colony_3
5	9/4 Pcon+pSB2K3 Colony_7	14	9/4 Pcon+pSB2K3 Colony_4
6	9/4 Pcon+pSB2K3 Colony_8	15	9/4 Pcon+pSB2K3 Colony_5
7	9/4 Pcon+pSB2K3 Colony_9	16	9/4 Pcon+pSB2K3 Colony_6
8	9/4 Pcon+pSB3T5 Colony_10	17	1kb ladder
9	9/4 pSB3T5(Nega) Colony_1

Lane	Colony PCR ProductsⅢ	Lane	Colony PCR ProductsⅢ
1	1kb ladder	10	9/4 pSB2K3(Nega) Colony_5
2	9/4 Pcon+pSB2K3 Colony_7	11	9/4 pSB1C3(Nega) Colony_1
3	9/4 Pcon+pSB2K3 Colony_8	12	9/4 pSB1C3(Nega) Colony_2
4	9/4 Pcon+pSB2K3 Colony_9	13	9/4 pSB1C3(Nega) Colony_3
5	9/4 Pcon+pSB2K3 Colony_10	14	9/4 pSB1C3(Nega) Colony_4
6	9/4 pSB2K3(Nega) Colony_1	15	9/4 pSB1C3(Nega) Colony_5
7	9/4 pSB2K3(Nega) Colony_2	16	9/4 pSB1C3(Nega) Colony_6
8	9/4 pSB2K3(Nega) Colony_3	17	1kb ladder
9	9/4 pSB2K3(Nega) Colony_4

Lane	Colony PCR ProductsⅣ	Lane	Colony PCR ProductsⅣ
1	1kb ladder	10	9/4 Pcon+pSB1C3 Colony_5
2	9/4 pSB1C3(Nega) Colony_7	11	9/4 Pcon+pSB1C3 Colony_6
3	9/4 pSB1C3(Nega) Colony_8	12	9/4 Pcon+pSB1C3 Colony_7
4	9/4 pSB1C3(Nega) Colony_9	13	9/4 Pcon+pSB1C3 Colony_8
5	9/4 pSB1C3(Nega) Colony_10	14	9/4 Pcon+pSB1C3 Colony_9
6	9/4 Pcon+pSB1C3 Colony_1	15	9/4 Pcon+pSB1C3 Colony_10
7	9/4 Pcon+pSB1C3 Colony_2	16	8/22 Miniprep BBa_I732095 Colony_3
8	9/4 Pcon+pSB1C3 Colony_3	17	1kb ladder
9	9/4 Pcon+pSB1C3 Colony_4

Lane	Colony PCR ProductsⅤ
1	1kb ladder
2	9/4 Pcon+pSB1C3 Colony_11
3	9/4 Negative Control
4	8/22 Miniprep BBa_I732095 Colony_3
5	-
6	-
7	-
8	-

Gel Extractions

No Name

Lane	Restriction Enzyme Digestion ProductⅠ	Volume/(µl)
1	1kb ladder	5
2	9/4 pSB2K3(E+P)	5
3	-	-
4	9/4 pSB2K3(E+P)	25
5	-	-
6	9/4 pSB2K3(Nega)	10

Lane	Restriction Enzyme Digestion ProductⅡ	Volume/(µl)
1	1kb ladder	5
2	9/4 pSB3T5(E+P)	5
3	-	-
4	9/4 pSB3T5(E+P)	25
5	-	-
6	9/4 pSB3T5(Nega)	10

Lane	Restriction Enzyme Digestion ProductⅢ	Volume/(µl)
1	1kb ladder	5
2	9/4 pSB4A5(E+P)	5
3	-	-
4	9/4 pSB4A5(E+P)	25
5	-	-
6	9/4 pSB4A5(Nega)	10

Lane	Restriction Enzyme Digestion ProductⅣ	Volume/(µl)
1	1kb ladder	5
2	-	-
3	-	-
4	9/4 pSB1C3(E+P)	10
5	-	-
6	-	-

DNA	Concentration/(µg/ml)	260/280	260/230
9/4 pSB2K3(E+P)	36	1.25	0.24
9/4 pSB3T5(E+P)	10	1.00	-/td>
9/4 pSB4A5(E+P)	40	-	0.01
9/4 pSB1C3(E+P)	75	1.35	0.14

9/8

Cloning PCR

Honda and Li

Target	mamB	mamQ	mamL
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5	12.5	12.5
Fw Primer/(µl)	1.5(mamB)	1.5(mamQ)	1.5(mamL)
Rv Primer/(µl)	1.5(mamB)	1.5(mamQ)	1.5(mamL)
AMB-1 Genome/(µl)	1	1	1
MilliQ/(µl)	10	10	10
TOTAL/(µl)	25	25	25

Steps	Temparature	Time	cycle
Pre Denature	98℃	5min	1time
Denature	98℃	20sec	25cycle
Annealing	65℃	15sec	25cycle
Extension	72℃	5min	25cycle
Final elongation	72℃	5min	1time
Final hold	4℃	-	-

Ligation

Honda

Insert/(µL)		Vector/(µL)		T4 Ligase/(µl)	2*Buffer/(µl)	MilliQ/(µl)	Total/(µl)
Pcon	1	pSB3T5	10	1	11	0	22
-(Negative Control)	0	pSB1C3	3	0	3	0	6

Electrophoresis

Li

Lane	Cloning PCR Products
1	1kb ladder
2	9/8 Cloning PCR (AMB-1 mamB)
3	9/8 Cloning PCR (AMB-1 mamQ)
4	9/8 Cloning PCR (AMB-1 mamL)
5	1kb ladder

Transformation

Honda

Sample/(µL)		CompetentCells/(µl)	Total/(µl)	Medium
9/8 Ligation Pcon+pSB3T5	3	30	33	LB(TET)
9/8 Ligation pSB3T5(Negative Control)	3	30	33	LB(TET)

Liquid Culture

No name

Name	medium
9/3 Transformation Pcon+pSB1C3 Colony_8	Plusgrow medium(CP)*2

9/9

DNA Purification

Li

DNA	Concentration/(µg/ml)	260/280	260/230
9/8 Cloning PCR (AMB-1 mamB)	165	1.54	1.57
9/8 Cloning PCR (AMB-1 mamQ)	250	1.69	1.92
9/8 Cloning PCR (AMB-1 mamL)	190	1.56	1.79

Miniprep

DNA	Concentration/(µg/ml)	260/280	260/230
9/8 Liquid Culture Pcon+pSB1C3	90	1.65	1.17

Colony PCR

Honda and Li

NameⅠ
-
9/8 Transformation Pcon+pSB3T5 Colony_1
9/8 Transformation Pcon+pSB3T5 Colony_2
9/8 Transformation Pcon+pSB3T5 Colony_3
9/8 Transformation Pcon+pSB3T5 Colony_4
9/8 Transformation Pcon+pSB3T5 Colony_5
9/8 Transformation pSB3T5(Nega) Colony_1
Negative control

Electrophoresis

Honda and Li

Lane	Cloning PCR Products
1	1kb ladder
2	9/8 Colony PCR Pcon+pSB3T5 Colony_1
3	9/8 Colony PCR Pcon+pSB3T5 Colony_2
4	9/8 Colony PCR Pcon+pSB3T5 Colony_3
5	9/8 Colony PCR Pcon+pSB3T5 Colony_4
6	9/8 Colony PCR Pcon+pSB3T5 Colony_5
7	9/8 Colony PCR pSB3T5(Nega) Colony_1
8	9/8 Colony PCR Nega

Restriction Enzyme Digestion

No name

Sample	DNA/(µl)	Pst1/(µl)	Spe1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/9 Miniprep Pcon+pSB1C3	22	1	1	3(H)	3	30
9/9 Miniprep Pcon+pSB1C3(Nega)	1	0	0	1(H)	8	10
8/2 Miniprep Pcon Colony_4	14	1	1	3(H)	11	30
8/2 Miniprep Pcon Colony_4(Nega)	1	0	0	1(H)	8	10

9/10

Gel Extractions

Tatsui and Yamaura

Lane	Sample	Volume/(µl)
1	1kb ladder	-
2	-	-
3	9/10 Digestion Pcon+pSB1C3(S+P)	-
4	-	-
5	9/10 Digestion Pcon+pSB1C3(Nega)	-
6	-	-
7	Pcon(X+P)	-
8	-	-
9	Pcon(Nega)	-
10	1kb ladder	-
11	-	-

DNAConcentration/(µg/ml)260/280260/230 Pcon(X+P)701.400.13

PCR

No Name

Target	E+X+RBS+mamB+S	X+RBS+mamL+His+S+P	X+RBS+mamQ+His+S+P
2×KAPA HiFi Hotstart Ready Mix/(µl)	12.5	12.5	12.5
Fw Primer/(µl)	0.75(E+X+RBS+mamB)	0.75(X+RBS+mamL)	0.75(X+RBS+mamQ)
Rv Primer/(µl)	0.75(S+mamB)	0.75(P+S+His+mamL)	0.75(P+S+His+mamQ)
9/9 DNA Purification Product/(µl)	Trace	Trace	Trace
MilliQ/(µl)	11	11	11
TOTAL/(µl)	25	25	25

Steps	Temparature	Time	cycle
Pre Denature	98℃	5min	1time
Denature	98℃	20sec	25cycle
Annealing	60℃	15sec	25cycle
Extension	72℃	15sec	25cycle
Final elongation	72℃	5min	1time
Final hold	12℃	-	-

Electrophoresis

Li

Lane	Cloning PCR Products
1	1kb ladder
2	9/10 PCR E+X+RBS+mamB+S(922bp)
3	9/10 PCR X+RBS+mamL+His+S+P(340bp)
4	-
5	1kb ladder
6	9/10 PCR X+RBS+mamQ+His+S+P(953bp)

DNA Purification

Honda and Yamaura

DNA	Concentration/(µg/ml)	260/280	260/230
9/10 PCR E+X+RBS+mamB+S	200	1.84	2.15
9/10 PCR X+RBS+mamL+His+S+P	120	1.69	1.84
9/10 PCR X+RBS+mamQ+His+S+P	200	1.70	2.16

Restriction Enzyme Digestion

Shimizu

Sample	DNA/(µl)	Xba1/(µl)	Pst1/(µl)	Buffur/(µl)	MilliQ/(µl)	TOTAL/(µl)
9/10 PCR E+X+RBS+mamB+S	10	1	1	3(H)	15	30
9/10 PCR X+RBS+mamL+His+S+P	16.7	1	1	3(H)	8.3	30
9/10 PCR X+RBS+mamQ+His+S+P	10	1	1	3(H)	15	30

@@ Line 228: / Line 228: @@
 <tr><td>Annealing</td><td>55℃</td><td>30sec</td><td>30cycle</td></tr>
 <tr><td>Extension</td><td>68℃</td><td>1min</td><td>30cycle</td></tr>
 </table>
@@ Line 696: / Line 697: @@
 <tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>25cycle</td></tr>
 <tr><td>Extension</td><td>72℃</td><td>5min</td><td>25cycle</td></tr>
+<tr><td>Final elongation</td><td>72℃</td><td>5min</td><td>1time</td></tr>
+<tr><td>Final hold</td><td>4℃</td><td>-</td><td>-</td></tr>
 </table>
@@ Line 775: / Line 778: @@
 <tr><td>6</td><td>9/8 Colony PCR Pcon+pSB3T5 Colony_5</td></tr>
 <tr><td>7</td><td>9/8 Colony PCR pSB3T5(Nega) Colony_1</td></tr>
-<tr><td>8</td>9/8 Colony PCR Nega<td></td></tr>
+<tr><td>8</td><td>9/8 Colony PCR Nega</td></tr>
 </table>
@@ Line 792: / Line 795: @@
 <h4>Gel Extractions</h4>
-<span class="author">Matsumoto</span>
+<span class="author">Tatsui and Yamaura</span>
 <table class="note">
 <tr><th>Lane</th><th>Sample</th><th>Volume/(&micro;l)</th></tr>
-<tr><td>1</td><td>9/1 pSB3T5 Colony_1 (Nega)</td><td>10</td></tr>
+<tr><td>1</td><td>1kb ladder</td><td>-</td></tr>
-<tr><td>2</td><td>9/1 pSB3T5 Colony_1 (E+P)(Confirmation)</td><td>5</td></tr>
+<tr><td>2</td><td>-</td><td>-</td></tr>
-<tr><td>3</td><td>9/1 pSB3T5 Colony_1 (E+P)(Extraction)</td><td>55</td></tr>
+<tr><td>3</td><td>9/10 Digestion Pcon+pSB1C3(S+P)</td><td>-</td></tr>
-<tr><td>4</td><td>1kb ladder</td><td>3</td></tr>
+<tr><td>4</td><td>-</td><td>-</td></tr>
-<tr><td>5</td><td>9/1 I732095 (Nega)</td><td>10</td></tr>
+<tr><td>5</td><td>9/10 Digestion Pcon+pSB1C3(Nega)</td><td>-</td></tr>
-<tr><td>6</td><td>9/1 I732095 (E+P)(Confirmation)</td><td>5</td></tr>
+<tr><td>6</td><td>-</td><td>-</td></tr>
-<tr><td>7</td><td>9/1 I732095 (E+P)(Extraction)</td><td>85</td></tr>
+<tr><td>7</td><td>Pcon(X+P)</td><td>-</td></tr>
-<tr><td>8</td><td>1kb ladder</td><td>3</td></tr>
+<tr><td>8</td><td>-</td><td>-</td></tr>
-<tr><td>9</td><td>9/1 pSB2K3 (Nega)</td><td>10</td></tr>
+<tr><td>9</td><td>Pcon(Nega)</td><td>-</td></tr>
-<tr><td>10</td><td>9/1 pSB2K3 (E+P)(Confirmation)</td><td>5</td></tr>
+<tr><td>10</td><td>1kb ladder</td><td>-</td></tr>
-<tr><td>11</td><td>9/1 pSB2K3 (E+P)(Extraction)</td><td>85</td></tr>
+<tr><td>11</td><td>-</td><td>-</td></tr>
 </table>
+<tr><th>DNA</th><th>Concentration/(&micro;g/ml)</th><th>260/280</th><th>260/230</th></tr>
+<tr><td>Pcon(X+P)</td><td>70</td><td>1.40</td><td>0.13</td></tr>
+</table>
+<h4>PCR</h4>
+<span class="author">No Name</span>
+<table class="note">
+<tr><th>Target</th><th>E+X+RBS+mamB+S</th><th>X+RBS+mamL+His+S+P</th><th>X+RBS+mamQ+His+S+P</th></tr>
+<tr><td>2×KAPA HiFi Hotstart Ready Mix/(&micro;l)</td><td>12.5</td><td>12.5</td><td>12.5</td></tr>
+<tr><td>Fw Primer/(&micro;l)</td><td>0.75(E+X+RBS+mamB)</td><td>0.75(X+RBS+mamL)</td><td>0.75(X+RBS+mamQ)</td></tr>
+<tr><td>Rv Primer/(&micro;l)</td><td>0.75(S+mamB)</td><td>0.75(P+S+His+mamL)</td><td>0.75(P+S+His+mamQ)</td></tr>
+<tr><td>9/9 DNA Purification Product/(&micro;l)</td><td>Trace</td><td>Trace</td><td>Trace</td></tr>
+<tr><td>MilliQ/(&micro;l)</td><td>11</td><td>11</td><td>11</td></tr>
+<tr><td>TOTAL/(&micro;l)</td><td>25</td><td>25</td><td>25</td></tr>
+</table>
+<table class="note">
+<tr><th>Steps</th><th>Temparature</th><th>Time</th><th>cycle</th></tr>
+<tr><td>Pre Denature</td><td>98℃</td><td>5min</td><td>1time</td></tr>
+<tr><td>Denature</td><td>98℃</td><td>20sec</td><td>25cycle</td></tr>
+<tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>25cycle</td></tr>
+<tr><td>Extension</td><td>72℃</td><td>15sec</td><td>25cycle</td></tr>
+<tr><td>Final elongation</td><td>72℃</td><td>5min</td><td>1time</td></tr>
+<tr><td>Final hold</td><td>12℃</td><td>-</td><td>-</td></tr>
+</table>
+<h4>Electrophoresis</h4>
+<span class="author">Li</span>
+<table class="note">
+<tr><th>Lane</th><th>Cloning PCR Products</td></tr>
+<tr><td>1</td><td>1kb ladder</td></tr>
+<tr><td>2</td><td>9/10 PCR E+X+RBS+mamB+S(922bp)</td></tr>
+<tr><td>3</td><td>9/10 PCR X+RBS+mamL+His+S+P(340bp)</td></tr>
+<tr><td>4</td><td>-</td></tr>
+<tr><td>5</td><td>1kb ladder</td></tr>
+<tr><td>6</td><td>9/10 PCR X+RBS+mamQ+His+S+P(953bp)</td></tr>
+</table>
+<h4>DNA Purification</h4>
+<span class="author">Honda and Yamaura</span>
+<table class="note">
+<tr><th>DNA</th><th>Concentration/(&micro;g/ml)</th><th>260/280</th><th>260/230</th></tr>
+<tr><td>9/10 PCR E+X+RBS+mamB+S</td><td>200</td><td>1.84</td><td>2.15</td></tr>
+<tr><td>9/10 PCR X+RBS+mamL+His+S+P</td><td>120</td><td>1.69</td><td>1.84</td></tr>
+<tr><td>9/10 PCR X+RBS+mamQ+His+S+P</td><td>200</td><td>1.70</td><td>2.16</td></tr>
+</table>
+<h4>Restriction Enzyme Digestion</h4>
+<span class="author">Shimizu</span>
+<table class="note">
+<tr><th>Sample</th><th>DNA/(&micro;l)</th><th>Xba1/(&micro;l)</th><th>Pst1/(&micro;l)</th><th>Buffur/(&micro;l)</th><th>MilliQ/(&micro;l)</th><th>TOTAL/(&micro;l)</tr>
+<tr><td>9/10 PCR E+X+RBS+mamB+S</td><td>10</td><td>1</td><td>1</td><td>3(H)</td><td>15</td><td>30</td></tr>
+<tr><td>9/10 PCR X+RBS+mamL+His+S+P</td><td>16.7</td><td>1</td><td>1</td><td>3(H)</td><td>8.3</td><td>30</td></tr>
+<tr><td>9/10 PCR X+RBS+mamQ+His+S+P</td><td>10</td><td>1</td><td>1</td><td>3(H)</td><td>15</td><td>30</td></tr>
+</table>
 <a name="0911" class="kyoto-jump"></a>

Team:Kyoto/Notebook

From 2014.igem.org

Revision as of 08:44, 26 September 2014

Notebook

Calendar

7/15

transformation

7/16

transformation

7/17

Colony PCR

Liquid Culture

7/18

Miniprep

7/30

Restriction Enzyme Digestion

Electrophoresis

7/31

Transformation

Liquid Culture

Restriction Enzyme Digestion

8/1

Electrophoresis

PCR

8/2

Miniprep

8/20

Transformation

8/21

Colony PCR

Electrophoresis

Liquid Culture

8/22

Miniprep

Restriction Enzyme Digestion

Transformation

8/25

Transformation

Liquid Culture

8/26

Miniprep

Liquid Culture

8/27

Miniprep

Transformation

8/28

Liquid Culture

8/29

Miniprep

Transformation

9/1

Colony PCR

Restriction Enzyme Digestion

Liquid Culture

9/2

Miniprep

Gel Extractions

9/3

Concentration Measurement

Ligation

Transformation

9/4

Colony PCR

Restriction Enzyme Digestion

9/5

Electrophoresis

Gel Extractions

9/8

Cloning PCR

Ligation

Electrophoresis

Transformation

Liquid Culture

9/9

DNA Purification

Miniprep

Colony PCR

Electrophoresis

Restriction Enzyme Digestion

9/10