Team:Kyoto/Notebook/DMS Synthesis

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Notebook -DMS-

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Note

2/10

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
J23100 5.7 - - 0.5 0.5 10xBuffer(K) 3 - 20.3 30
RBS-GFP-DT 7.5 - 0.5 - 0.5 10xBuffer(M) 3 3 15.5 30

Electrophoresis/Gel Extraction

Yasuda
Lane Sample
1 1kbp ladder(2µL)
2 J23100 DNA control
3 J23100 S,P experimental
4 J23100 S,P experimental
Lane Sample
1 1kbp ladder(2µL)
2 RBS-GFP-DT DNA control
3 RBS-GFP-DT X,P experimental
4 RBS-GFP-DT X,P experimental

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
J23100 10 - - 0.5 0.5 10xBuffer(K) 3 - 16 30

2/12

Electrophoresis/Gel Extraction

Yasuda
Lane Sample
1 1kbp ladder(2µL)
2 J23100 DNA control
3 J23100 S,P experimental
4 J23100 S,P experimental
Sample Concentration/(µg/mL) 260/280 260/230
GFP 16.7 1.46 0.64
Pcon 17.2 1.69 0.26

Ligation

Inoue
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
2/10 J23100 S,P 2100bp 5.8 2/10 RBS-GFP-DT 898bp 4.9 2x Buffer 5 T4 Ligase 3 - 10
　　　　　　　

Vector and insert are evaporated

Transformation

Yasuda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
2/12 J23119(A) Pcon 1 Self-Making Competent Cells (DH5α) 10 11 SOC
2/12 I712074 (Ak) T7 1 Self-Making Competent Cells (DH5α) 10 11 SOC
2/12 Ligation Pcon+RBS-GFP-PT 1 Self-Making Competent Cells (DH5α) 10 11 SOC

2/13

Colony PCR

Yasuda,Murakami
Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
30 1.2 1.2 27.6 60
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
5min 30sec 30sec 72sec

Denature-Annealing-Extension 30cycle

Number Sample
1 Pcon-RBS-GFP-DT (1)
2 Pcon-RBS-GFP-DT (2)
3 Pcon-RBS-GFP-DT (3)
4 Pcon-RBS-GFP-DT (4)
5 T7

Electrophoresis

Honda
Lane Sample
1 1kbp ladder
2 2/12 Pcon-RBS-GFP-DT (1)
3 2/12 Pcon-RBS-GFP-DT (2)
4 2/12 Pcon-RBS-GFP-DT (3)
5 2/12 Pcon-RBS-GFP-DT (4)
6 2/12 I712074(Ak) T7

Liquid Culture

No Name
Sample Medium
Pcon-RBS-GFP-DT Amp

2/14

Miniprep

Murakami
Sample
2/13 Pcon-RBS-GFP-DT(1)
2/13 Pcon-RBS-GFP-DT(2)

2/23

Self-Making Competent Cells (DH5α)

Tatsui and Kojima

3/4

Restriction Enzyme Digestion

Kato, Murata and Shimazaki
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
9/8 J23100 325µg/ml 6 - - 0.5 0.5 H 3 3 17 30
9/8 J23100 325µg/ml control 0.3 - - - - H 1 1 7.7 10
9/28 RBS-GFP-DT 215.9µg/ml 9.3 - 0.5 - 0.5 H 3 3 13.7 30
control 0.5 - - - - H 1 1 7.5 10

Electrophoresis

Kato, Murata and Shimazaki
Lane Sample
1 1kbp ladder
2 9/8 J23100 S,P
3 9/8 J23100 control
4 9/28 RBS-GFP DT X,P
5 9/28 RBS-GFP DT control

Gel Extraction

Kato, Murata and Shimazaki
Lane Sample
1 9/8 J23100 S,P
2 Blank
3 Blank
4 9/28 RBS-GFP-DT X,P
5 1kbp ladder

3/5

Ligation

Kato, Shimazaki and Murata
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
3/4 J23100 S,P 4.8 3/4 RBS-GFP-DT X,P 9.2 Reaction Buffer 2 Ligase(Bio Lub) 1 3 20

Transformation

No Name
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
3/5 J23119 1 Self-Making Competent Cells (DH5α) 10 11 SOC
3/5 I712074 1 Self-Making Competent Cells (DH5α) 10 11 SOC
3/5 ligation Pcon RBS-GFP-DT 1 Self-Making Competent Cells (DH5α) 10 11 SOC

3/6

Colony PCR

Kato and Shimazaki
Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
65 2.6 2.6 59.8 130
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
5min 30sec 30sec 72sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/5 J23119 (1)
2 3/5 J23119 (2)
3 3/5 J23119 (3)
4 3/5 J23119 (4)
5 3/5 I712074 (1)
6 3/5 I712074 (2)
7 3/5 I712074 (3)
8 3/5 I712074 (4)
Number Sample
1 3/5 Pcon RBS-GFP-DT (1)
2 3/5 Pcon RBS-GFP-DT (2)
3 3/5 Pcon RBS-GFP-DT (3)
4 3/5 Pcon RBS-GFP-DT (4)

Electrophoresis

Murata
Lane Sample
1 1kbp ladder
2 3/5 J23119 (1)
3 3/5 J23119 (2)
4 3/5 J23119 (3)
5 3/5 J23119 (4)
6 3/5 I712074 (1)
7 3/5 I712074 (2)
8 3/5 I712074 (3)
9 3/5 I712074 (4)
10 3/5 Pcon RBS-GFP-DT (1)
11 3/5 Pcon RBS-GFP-DT (2)
12 3/5 Pcon RBS-GFP-DT (3)
13 3/5 Pcon RBS-GFP-DT (4)
14 1kbp ladder

3/7

Miniprep

Murata
Sample
3/6 Pcon RBS-GFP-DT (1)
3/6 Pcon RBS-GFP-DT (2)

3/10

Transformation

Okazaki
Sample/(µL) CompetentCells/(µL) Medium
3/10 RBS-GFP Self-Making Competent Cells (DH5α) CP
RBS Self-Making Competent Cells (DH5α) Amp
DT Self-Making Competent Cells (DH5α) CP
3/10 T7 Self-Making Competent Cells (DH5α) Amp

3/11

Colony PCR

Shimazaki

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
50 2 2 46 100
Predenature Denature Annealing Extension
97°C 94°C 55°C 68°C
5min 30sec 30sec 60sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/10 RBS (1)
2 3/10 RBS (2)
3 3/10 pSB1C3 (1)
4 3/10 pSB1C3 (2)
5 3/10 DT (1)
6 3/10 DT (2)
7 3/10 T7 (1)
8 3/10 T7 (2)
9 control

3/12

Electrophoresis

Shimazaki
Lane Sample
1 3/10 RBS (1)
2 3/10 RBS (2)
3 3/10 pSB1C3 (1)
4 3/10 pSB1C3 (2)
5 3/10 DT (1)
6 3/10 DT (2)
7 3/10 T7 (1)
8 3/10 T7 (2)
9 control
10 1kbp ladder

Colony PCR

Shimazaki

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
50 2.0 2.0 46 100
Predenature Denature Annealing Extension
97°C 94°C 55°C 68°C
5min 30sec 30sec 60sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/10 RBS 3
2 3/10 RBS 4
3 3/10 pSB1C3 4
4 3/10 pSB1C3 5
5 3/10 DT 4
6 3/10 DT 5
7 3/10 T7 3
8 3/10 T7 4
9 new QuickTaq control
10 old QuickTaq control

3/14

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 3/10 RBS (1)
3 3/10 RBS (2)
4 3/10 pSB1C3 (3)
5 3/10 pSB1C3 (4)
6 3/10 DT (5)
7 3/10 DT (6)
8 3/10 T7 (7)
9 3/10 T7 (8)
10 old Quick Taq control (9)
11 new Quick Taq control (10)
12 1kbp ladder

Colony PCR

Kojima

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
17.5 0.7 0.7 16.1 35
Predenature Denature Annealing Extension
97°C 95°C 58°C 68°C
5min 30sec 30sec 30sec

Denature-Annealing-Extension 30cycle

Number Sample
1 RBS-4
2 DT-5
3 control

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 RBS-4
3 DT-5
4 control

Colony PCR

Yasuda and Shimazaki

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
17.5 0.7 0.7 16.1 35
Predenature Denature Annealing Extension
97°C 95°C 58°C 68°C
5min 30sec 30sec 60sec

Denature-Annealing-Extension 30cycle

Number Sample
1 pSB1C3-3
2 T7-5
3 control

Electrophoresis

Shimazaki
Lane Sample
1 1kbp ladder
2 pSB1C3-3
3 T7-5
4 control
4 2/10 MilliQ

3/15

Liquid Culture

Yasuda and Murata
Sample Medium
Ruegeria pomeroyi fungus liquid YTSS medium

3/17

Liquid Culture

Yoshida
Sample Medium
3/10 pSB1C3-3 CP
3/10 DT-5 CP
3/10 T7-5 Amp
3/10 RBS-4 Amp

3/18

Miniprep

Murata and Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
3/17 DT CP 64.4 1.17 0.29
3/17 RBS Amp 61.4 1.28 0.36
3/17 T7 Amp 66.8 1.22 0.34
3/17 pSB1C3 64.2 1.31 0.39

DNA Extraction

Yasuda and Murata

Nuelei Lysis Solution Solution 600µL pipeting 85°C 5min incuvate and cooling

RNase Solution 3µL invert(2-5times) 37°C 45min incuvate and cooling

Protein Preciprtation Solution 200µL Highspeed voltex on ice 5min and 16000g 3min (Supernatant ladled)

isopropyl alcohol 600µL invert 16000g 2min (Supernatant discarded)

70%ethanol 600µL invert 16000g 2min aspirate　10-15min air-drying

DNA Rehydration Solution 100µL 65°C 1h incubate

Restriction Enzyme Digestion

Yoshida
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 E,S 3/18 miniprep product 20 1 1 - - M 3 3 2 30
control 3 - - - - M 1 1 5 10

Cloning PCR

Murata and Yasuda
2K KAPA HiFi HotStart ReadyMix/(µL) F dddD 66/(µL) R dddD 65/(µL) R.pomeroyi Genome/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 3 8 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 65°C 72°C 72°C
3min 20sec 15sec 150sec 1sec

Denature-Annealing-Extension 25cycle

3/19

　　　　　

Electrophoresis

Yoshida
Lane Sample
1 1kbp ladder
2 pSB1C3 E,X
3 pSB1C3 control

Restriction Enzyme Digestion

Yoshida
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 3/18 miniprep product 20 1 - 1 - H 3 - 5 30
control 3 - - - - H 1 - 6 10

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 dddD PCR product

PCR

Kojima
3/19 DNA Purification product/(µL) Fw/(µL) Rv/(µL) HiFi Mix/(µL) MilliQ/(µL) Total/(µL)
1 0.75 0.75 12.5 10 25
R.pomeroyi Genome/(µL) Fw/(µL) Rv/(µL) HiFi Mix/(µL) MilliQ/(µL) Total/(µL)
3 0.75 0.75 12.5 8 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 68°C 72°C 72°C
5min 20sec 15sec 90sec 5min

Denature-Annealing-Extension 25cycle

3/20

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 pSB1C3 E,S
3 control
4 dddD(1) 3/19 PCR product
5 dddD(2) 3/19 PCR product

Gel Extraction

Yasuda and Tatsui
Lane Sample
1 Blank
2 1kbp ladder
3 pSB1C3
4 Blank
5 pSB1C3

Gel Extraction

Murara
Lane Sample
1 1kbp ladder
2 dddD
3 dddD
Sample Concentration/(µg/mL) 260/280 260/230
dddD 35 1.63 0.85

3/24

Restriction Enzyme Digestion

Honda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 11 0.5 0.5 - - M 1.5 1.5 - 15
dddD 12.5 0.5 - 0.5 - M 1.5 - - 15

PCR

Honda
2x KAPA HiFi HotStart ReadyMix/(µL) F dddD 62/(µL) R dddD 68/(µL) Templete 3/24 dddD/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 pick up 11 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 68°C 72°C 72°C
5min 20sec 15sec 90sec 5min

Denature-Annealing-Extension 25cycle

Number Sample
1 dddD
Sample Concentration/(µg/mL) 260/280 260/230
dddD 62.7 1.77 1.88

Ligation

Honda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
dddD 1.3 pSB1C3 0.43 2xBuffer 5 T4Ligase 1 2.27 10

Liquid Culture

Tatsui
Sample Medium
pSB1C3(3) CP
pSB1C3(4) CP

3/25

Miniprep

Tatsui
Sample Concentration/(µg/mL)
pSB1C3 3/24 (3) 41
pSB1C3 3/24 (4) 57

Restriction Enzyme Digestion

Murata
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 3/24 (3) 15.4 0.5 - 0.5 - H 3 - 10.6 30
3/24 PCR product dddD E,S 3.2 0.5 - 0.5 - H 3 - 22.8 30

Gel Extraction

Murata
Lane Sample
1 1kbp ladder
2 pSB1C3(3) E,S 3/25
3 Blank
4 Blank
5 Blank
6 dddD 3/25 E,S
7 dddD 3/25 E,S
Sample Concentration/(µg/mL) 260/280 260/230
dddD 4.9 17.5 0.52
pSB1C3 2.0 - 0.21

3/26

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 2047bp 30 dddD 2545bp 30 2x Rapid Ligation Buffer 5 T4Ligase 3 - 10

Vector and insert are elaborated.

Transformation

Yasuda
Sample/(µL)
dddD(pSB1C3)

3/27

Restriction Enzyme Digestion

Murata
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3(4) 3/25 20.1 0.5 - 0.5 - 10xBuffer 3 - 5.9 30
dddD 3/24 PCR product 3.2 0.5 - 0.5 - 10xBuffer 3 - 22.8 30

Gel Extraction

Honda
Lane Sample
1 1kbp ladder
2 pSB1C3 E,S 3/27
3 pSB1C3 E,S 3/27
4 Blank
5 dddD E,S 3/27
6 dddD E,S 3/27
Sample Concentration/(µg/mL) 260/280 260/230
pSB1C3 9 1.58 0.04
dddD 16.0 1.78 0.08

Ligation

Honda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 11 dddD 15 2x Rapid Ligation Buffer 5 T4 DNA Ligase 1 4 10

3/28

PCR

Yasuda
2x KAPA HiFi HotStart ReadyMix/(µL) F Sequence primer/(µL) R Spe1-dddD/(µL) Templete/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 pick up 11 25
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 68°C 72°C 72°C
5min 20sec 15sec 90sec 5min

Denature-Annealing-Extension 25cycle

Number Sample
1 Fw:Sequence primer (1) 57.9/Rv:Spe1 dddD 2276bp
2 Fw:Sequence primer (2) 57.3/Rv:Spe1 dddD 1907bp
3 Fw:Sequence primer (3) 57.6/Rv:Spe1 dddD 1527bp
4 Fw:Sequence primer (4) 56.3/Rv:Spe1 dddD 1189bp
5 Fw:Sequence primer (5) 57.2/Rv:Spe1 dddD 799bp
6 Fw:Sequence primer (6) 56.4/Rv:Spe1 dddD 398bp
7 Fw:Sequence primer VF2 56.4/Rv:Spe1 dddD 266bp
8 Fw:pre-dddD PCR product 62/Rv:Spe1 dddD
9 Fw:Blank/Rv:Spe1 dddD

Electrophoresis

Murata
Lane Sample
1 1kbp ladder
2 Seaquence primer (1) PCR product
3 Seaquence primer (2) PCR product
4 Seaquence primer (3) PCR product
5 Seaquence primer (4) PCR product
6 Seaquence primer (5) PCR product
7 Seaquence primer (6) PCR product
8 Seaquence primer VF2
9 F pre-dddD cloning
10 Blank
11 1kbp ladder

Transformation

Kojima
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
dddD 1 Self-Making Competent Cells (DH5α) 10 11 CP

3/30

Colony PCR

Yasuda
Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension
97°C 95°C 58°C 68°C
5min 30sec 30sec 168sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/26 dddD(pSB1C3) (1)
2 3/26 dddD(pSB1C3) (2)
3 3/26 dddD(pSB1C3) (3)
4 3/26 dddD(pSB1C3) (4)
5 3/26 dddD(pSB1C3) (5)
6 3/28 dddD(pSB1C3) (6)
7 3/28 dddD(pSB1C3) (7)
8 3/28 dddD(pSB1C3) (8)
9 3/28 dddD(pSB1C3) (9)
10 3/28 dddD(pSB1C3) (10)

3/31

Electrophoresis

Yasuda
Number Sample
1 1kbp ladder
2 3/26 dddD(pSB1C3) (1)
3 3/26 dddD(pSB1C3) (2)
4 3/26 dddD(pSB1C3) (3)
5 3/26 dddD(pSB1C3) (4)
6 3/26 dddD(pSB1C3) (5)
7 1kbp ladder
8 3/28 dddD(pSB1C3) (6)
9 3/28 dddD(pSB1C3) (7)
10 3/28 dddD(pSB1C3) (8)
11 3/28 dddD(pSB1C3) (9)
12 3/28 dddD(pSB1C3) (10)
13 1kbp ladder

7/31

Restriction Enzyme Digestion

Nakamura and Matsumoto
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
3/24 dddD PCR product 3.2 1 - 1 - H 3 - 21.8 30
pSB1C3 18.5 1 - 1 - H 3 - 6.5 30
pSB1C3 control 6.2 - - - - H 1 - 2.8 10

8/1

Gel Extraction

Murata and Yasuda
Lane Sample
1 1kbp ladder
2 3/24 dddD PCR product E,S
3 3/24 dddD PCR product E,S
4 Blank
5 pSB1C3 E,S
6 pSB1C3 E,S
7 Blank
8 1kbp ladder
9 3/24 dddD PCR product control
10 pSB1C3 control
Sample Concentration/(µg/mL) 260/280 260/230
pSB1C3 13.3 1.90 -
dddD 10.8 1.64 0.15

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
8/1 pSB1C3 gel extraction product 2047bp 1.5 8/1 dddD gel extraction product 2545bp 6.9 10xT4Ligase 2 T4Ligase 1 8.6 20

Transformation

Murata
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
pSB1C3-dddD 3 Self-Making Competent Cells (DH5α) 30 33 SOC

8/2

PCR Cloning

Murata
dddD without Pre-Suf 2x KAPA HiFi Hotstart Ready Mix/(µL) F Pre-dddD PCR product 62/(µL) Rv Spe1-dddD PCR product 68/(µL) MilliQ/(µL) Total/(µL)
pick up 12.5 0.75 0.75 11 25
Predenature Denature Annealing Extension
95°C 98°C 65°C 72°C
5min 20sec 15sec 50sec

Denature-Annealing-Extension 25cycle

DNA Purification

Murata

8/2 Pre-dddD-Suf PCR product

Colony PCR

Honda,Nakamura and Matsumoto

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
15 0.6 0.6 13.8 30
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Number Sample
1 pSB1C3-dddD(1)
2 pSB1C3-dddD(2)
3 pSB1C3-dddD(3)

Concentration Measurement

Nakamura and Matsumoto
Sample Concentration/(µg/mL) 260/280 260/230
dddD PCR product 245 1.59 2.17

Restriction Enzyme Digestion

Nakamura and Matsumoto
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
8/2 dddD PCR product 8.2 1 - 1 - H 3 - 16.8 30
8/2 dddD PCR product control 0.41 - - - - H 1 - 8.6 10
pSB1C3 12.7 1 - 1 - H 3 - 12.3 30
pSB1C3 control 0.04 - - - - H 1 - 8.4 10

8/3

Electrophoresis

Murata
Lane Sample
1 pSB1C3-dddD(1)
2 pSB1C3-dddD(2)
3 pSB1C3-dddD(3)
4 1kb ladder (3µL)
5 1kb ladder (1µL)

Gel Extraction

Tatsui
Lane Sample
1 1kb ladder
2 Blank
3 pSB1C3 2070bp
4 Blank
5 pSB1C3
6 Blank
7 8/2 dddD PCR product 2544bp
8 Blank
9 8/2 dddD PCR product
Sample Concentration/(µg/mL) 260/280 260/230
pSB1C3 12.5 1.37 0.42
dddD PCR product 35 1.54 0.85

Ligation

no name
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 7 dddD 7 2x Buffer 15 T4 Ligase 1 - 30
pSB1C3 7 dddD 14 2x Buffer 22 T4 Ligase 1 - 44
pSB1C3 7 dddD - 2x Buffer 15 T4 Ligase 1 7 30

Transformation

no name
Sample CompetentCells Medium
(1) Self-Making Competent Cells (DH5α) CP
(2) Self-Making Competent Cells (DH5α) CP

8/4

Colony PCR

Honda

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
40 1.6 1.6 36.8 80
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Lane Sample
1 dddD+pSB1C3(1)-1
2 dddD+pSB1C3(1)-2
3 dddD+pSB1C3(2)-1
4 dddD+pSB1C3(2)-2
5 dddD+pSB1C3(2)-3
6 dddD+pSB1C3(2)-4
7 pSB1C3 control-1
8 control

Electrophoresis

Matsumoto
Lane Sample
1 1kbp ladder
2 dddD+pSB1C3(1)-1
3 dddD+pSB1C3(1)-2
4 dddD+pSB1C3(2)-1
5 dddD+pSB1C3(2)-2
6 dddD+pSB1C3(2)-3
7 dddD+pSB1C3(2)-4
8 pSB1C3 control-1
9 control
10 1kbp ladder

PCR

Honda

Unexpected bands are confirmed in former Electrophoresis, so contamination of MilliQ or Quicktaq is suspected. For confirming contamination, PCR and Electrophoresis are conducted.

* suspicious samples for contamination

(1)

*Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10

(2)

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10

(3)

Quick Taq/(µL) VF2/(µL) VR/(µL) *MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10

(4)

*Quick Taq/(µL) VF2/(µL) VR/(µL) *MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Honda
Lane Sample
1 1kbp ladder
2 (1)
3 (2)
4 (3)
5 (4)

8/18

PCR

Honda

For confirming contamination of VF2 and VR primers, we pour samples into new tubes and perform PCR and Electrophoresis.

(1)

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10

control(2)

Quick Taq/(µL) VF2/(µL) VR/(µL) 8/2 Pcon3/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 0.6 4.6 10

(3)

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10

(4)

Quick Taq/(µL) VF2/(µL) VR/(µL) 8/2 Pcon3/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 0.6 4.0 10
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Honda
Number Sample
1 1kbp ladder
2 (1)
3 (2)
4 (3)
5 (4)

Restriction Enzyme Digestion

Honda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
dddD 8/2 PCR product 5.5 0.7 - 0.7 - H 2 - 11.1 20
dddD 8/2 PCR product control 0.41 - - - - H 1 - 8.6 10
pSB1C3 12.7 1 - 1 - H 3 - 8.4 10
pSB1C3 control 0.64 - - - - H 1 - 8.4 10

Colony PCR

Honda

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
40 1.6 1.6 36.8 80
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Number Sample
1 dddD+pSB1C3(1)-1
2 dddD+pSB1C3(1)-2
3 dddD+pSB1C3(2)-1
4 dddD+pSB1C3(2)-2
5 dddD+pSB1C3(2)-3
6 dddD+pSB1C3(2)-4
7 pSB1C3 control-1
8 control

Electrophoresis

Honda
Lane Sample
1 1kbp ladder
2 dddD+pSB1C3(1)-1
3 dddD+pSB1C3(1)-2
4 dddD+pSB1C3(2)-1
5 dddD+pSB1C3(2)-2
6 dddD+pSB1C3(2)-3
7 dddD+pSB1C3(2)-4
8 pSB1C3 control-1
9 control

Gel Extraction

Murata
Lane Sample
1 1kbp ladder
2 dddD control
3 dddD E,S
4 Blank
5 pSB1C3 control
6 pSB1C3 E,S
Sample Concentration/(µg/mL) 260/280 260/230
8/18 pSB1C3 105 1.30 0.85
8/18 dddD 105 1.27 0.73

8/19

Ligation

Honda,Li,Yamauchi

(1)

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 3.6 dddD 1 2xBuffer 5.6 T4Ligase 1 - 11.2

(2)

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 3.6 - - 2xBuffer 5.6 T4Ligase 1 1 11.2

8/20

Transformation

Murata
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
8/19 Ligation (1) 11.2 Competent Cells (DH5α) 50 61.2 SOC
8/19 Ligation (2) 11.2 Competent Cells (DH5α) 50 61.2 SOC

Ligation

Nakamura,Honda

(1)

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 10.8 dddD 3 2xBuffer 16.8 T4Ligase 3 - 33.6

(2)

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 control 10.8 - - 2xBuffer 16.8 T4Ligase 3 3 33.6

Transformation

Nakamura,Honda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
dddD+pSB1C3 5 Competent Cells (DH5α) 20 25 SOC
control 5 Competent Cells (DH5α) 20 25 SOC

8/21

Colony PCR

Honda,Nakamura

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
35 1.4 1.4 32.2 70
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Nakamura
Number Sample
1 8/20 dddD+pSB1C3(1)-1
2 8/20 dddD+pSB1C3(1)-2
3 8/20 dddD+pSB1C3(2)-1
4 8/20 dddD+pSB1C3(2)-2
5 8/20 dddD+pSB1C3(3)-1
6 8/20 dddD+pSB1C3(3)-2
7 control
Number Sample
1 1kbp ladder
2 8/20 dddD+pSB1C3(1)-1
3 8/20 dddD+pSB1C3(1)-2
4 8/20 dddD+pSB1C3(2)-1
5 8/20 dddD+pSB1C3(2)-2
6 8/20 dddD+pSB1C3(3)-1
7 8/20 dddD+pSB1C3(3)-2
8 control

Gel Extraction

Yasuda
Lane Sample
1 1kbp ladder
2 8/22 Mp(1) E,S
3 8/22 Mp(1) E,S
4 8/22 Mp(1) E,S
Lane Sample
1 1kbp ladder
2 8/22 Mp(2) E,S
3 8/22 Mp(2) E,S
4 Blank
5 1kbp ladder
6 8/22 Mp(3) E,S
7 8/22 Mp(3) E,S

8/25

Gel Extraction

Yasuda
Lane Sample
1 1kbp ladder
2 8/22 Mp(1) E,S
3 8/22 Mp(1) E,S
4 8/22 Mp(1) E,S

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
8/22 Mp(1) E,S 1.3 8/18 dddD gel extraction product 0.71 2x Buffer 5 T4 Ligase 0.3 2.69 10
8/22 Mp(2) E+S 0.95 8/18 dddD gel extraction product 0.71 2x Buffer 5 T4 Ligase 0.3 3.04 10
8/22 Mp(3) E+S 0.69 8/18 dddD gel extraction product 0.71 2x Buffer 5 T4Ligase 0.3 3.3 10

8/26

Transformation

Nakamura,Matsumoto
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
8/25 Ligation (1) 10 Competent Cells (DH5α) 50 60 SOC
8/25 Ligation(2) 10 Competent Cells (DH5α) 50 60 SOC
8/25 Ligation(3) 10 Competent Cells (DH5α) 30 40 SOC

8/27

Colony PCR

Murata

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
80 3.2 3.2 73.6 160
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 140sec

Denature-Annealing-Extension 30cycle

Number Sample
1 8/26 dddD+pSB1C3(1)-1
2 8/26 dddD+pSB1C3(1)-2
3 8/26 dddD+pSB1C3(1)-3
4 8/26 dddD+pSB1C3(1)-4
5 8/26 dddD+pSB1C3(1)-5
6 8/26 dddD+pSB1C3(2)-1
7 8/26 dddD+pSB1C3(2)-2
8 8/26 dddD+pSB1C3(2)-3
9 8/26 dddD+pSB1C3(2)-4
10 8/26 dddD+pSB1C3(2)-5
11 8/26 dddD+pSB1C3(3)-1
12 8/26 dddD+pSB1C3(3)-2
13 8/26 dddD+pSB1C3(3)-3
14 8/26 dddD+pSB1C3(3)-4
15 8/26 dddD+pSB1C3(3)-5
16 control

Electrophoresis

Yasuda
Lane Sample
1 1kbp ladder
2 8/26 dddD+pSB1C3(1)-1
3 8/26 dddD+pSB1C3(1)-2
4 8/26 dddD+pSB1C3(1)-3
5 8/26 dddD+pSB1C3(1)-4
6 8/26 dddD+pSB1C3(1)-5
Lane Sample
1 1kbp ladder
2 8/26 dddD+pSB1C3(2)-1
3 8/26 dddD+pSB1C3(2)-2
4 8/26 dddD+pSB1C3(2)-3
5 8/26 dddD+pSB1C3(2)-4
6 8/26 dddD+pSB1C3(2)-5
7 1kbp ladder
8 8/26 dddD+pSB1C3(3)-1
9 8/26 dddD+pSB1C3(3)-2
10 8/26 dddD+pSB1C3(3)-3
11 8/26 dddD+pSB1C3(3)-4
12 8/26 dddD+pSB1C3(3)-5
13 1kbp ladder
14 control

8/29

PCR

Matsumoto

Master Mix

2x KAPA HiFi HotStart ReadyMix /(µL) V Pre-dddD 62/(µL) Rv Spe1-dddD 68/(µL) MilliQ/(µL) DNA Purification dddD without Pre-Suf Total/(µL)
12.5 0.75 0.75 11 pick up 25
Predenature Denature Annealing Extension
95°C 98°C 65°C 72°C
5min 20sec 15sec 50sec

Denature-Annealing-Extension 25cycle

DNA Purification

Matsumoto
Sample Concentration/(µg/mL) 260/280 260/230
8/29 Pre-dddD-Suf PCR product 335 1.73 1.91

Restriction Enzyme Digestion

Matsumoto
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
8/29 Pre-dddD-Suf PCR product 6.0 1 - 1 - H 3 - 19 30

DNA Purification

Matsumoto
Sample Concentration/(µg/mL) 260/280 260/230
8/29 Pre-dddD-Suf Restriction Enzyme Digestion product 60 1.64 0.94

Ligation

Matsumoto
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 E,S gel extraction product 4.7 dddD-pSB1C3 4 2x Buffer 9.7 T4 Ligase 1 - 19.4
pSB1C3 E,S gel extraction product control 4.7 - - 2x Buffer 9.7 T4 Ligase 1 4 19.4

Transformation

Matsumoto
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
dddD 8/29 ligation product 10 DH5α 40 50 SOC
8/29 ligation product control 10 DH5α 40 50 SOC
pSB1C3 control 1 DH5α 20 21 SOC

9/1

Colony PCR

Honda

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
30 1.2 1.2 27.6 60
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Number Sample
1 pSB1C3-dddD(1)
2 pSB1C3-dddD(2)
3 pSB1C3-dddD(3)
4 pSB4A5-(1)
5 pSB4A5-(2)
6 control

Electrophoresis

Matsumoto
LaneSample
11kb ladder
29/1 dddD+pSB1C3-1
39/1 dddD+pSB1C3-2
49/1 dddD+pSB1C3-3
59/1 pSB4A5-1
69/1 pSB4A5-2
7control
81kb ladder

Colony PCR

Matsumoto

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
30 1.2 1.2 27.6 60
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Number Sample
1 pSB1C3-dddD(1)
2 pSB1C3-dddD(2)
3 pSB1C3-dddD(3)
4 pSB4A5-(1)
5 pSB4A5-(2)
6 control

Electrophoresis

Matsumoto
LaneSample
11kb ladder
29/1 dddD+pSB1C3-1
39/1 dddD+pSB1C3-2
49/1 dddD+pSB1C3-3
59/1 pSB4A5-1
69/1 pSB4A5-2
7control
81kb ladder

9/2

Electrophoresis

Mastumoto
LaneSample
11kb ladder
28/29 dddD(DNA purification product)1µL+MilliQ 5µL+looding dye(x6)1µL
3control

Cloning

Yasuda
2x KAPA HiFi HotStart ReadyMix/(µL) Fw REDOX cloning primer 59/(µL) Rv REDOX primer Ver.2 64/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 65°C 72°C 72°C 4°C
5min 20sec 15sec 2min10sec 5min Infinity

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Matsumoto
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
8/29 dddD(DNA purification product)61-1--3-1930

Cloning

Yasuda

Master Mix(x4)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw primer 59/(µL) Rv primer Ver.2 64/(µL) 3/19 dddD(DNA purification) /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 0.1 11 25
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 65°C 72°C 72°C 4°C
5min 20sec 15sec 2min10sec 5min Infinity

Denature-Annealing-Extension 25cycle

9/3

Gel Extraction

Yamauchi
LaneSample
2dddD (E,S) 2544bp.
4dddD (E,S)
61kb ladder

DNA Purification

Matsumoto
SampleConcentration/(µg/mL)260/280260/230
Pre-dddD-suf6851.662.13

Ligation

Honda, Li and Yamauchi

(1)insert: dddD

Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
pSB1C31.5dddD6.32x Buffer8.8T4 Ligase1-17.6

(2)control

Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
pSB1C31.5dddD-2x Buffer8.8T4 Ligase16.317.6

Transformation

Yamauchi
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/3 Ligation product (dddD)3DH5α1518SOC
9/3 Ligation product (control)2DH5α1012SOC

30min on ice
1min heat shock
2min on ice

(1)Adding SOC medium(4 times) to each of them and doing preculture at 37°C for 1 hour.
(2)Adding SOC medium to each of them, up to 100µL.

9/4

Electrophoresis

Honda
LaneSample
11kb ladder
29/3 Ligation dddD + pSB1C3
38/20 Ligation dddD + pSB1C3
48/1 Ligation dddD + pSB1C3
59/3 dddD (Gel extraction product)(E,P)
69/3 dddD (DNA purification product)
71kb ladder

Cloning

Murata and Honda

(1)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw primer AT cloning primer 61/(µL) Rv primer AT cloning primer 61/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
(2)
2x KAPA HiFi HotStart ReadyMix/(µL) Fw SAMmt cloning primer 58/(µL) Rv SAMmt cloning primer 57/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
(3)
2x KAPA HiFi HotStart ReadyMix/(µL) Fw DECARB cloning primer 56/(µL) Rv DECARB cloning primer 56/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
(4)
2x KAPA HiFi HotStart ReadyMix/(µL) Fw DiDECARB cloning primer 57/(µL) Rv DiDECARB cloning primer 57/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(1)(4)

Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

(2)(3)
Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 70sec 5min Infinity

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda
LaneSample
11kb ladder
2AT cloning(1)
3SAMmt cloning(2)
4DECARB cloning(3)
5DiDECARB cloning(4)
61kb ladder

Restriction Enzyme Digestion

Yasuda

(1)

Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
I732095 8/22 miniprep (2)(255ng/µL)7.81-1-H(x10)3-17.230
I732095 8/22 miniprep (2)(255ng/µL)0.4----H(x10)1-8.610

incubate at 37°C overnight

(2)

Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
I732095 8/22 miniprep (2)(255ng/µL)7.81-1-H(x10)3-17.230
I732095 8/22 miniprep (2)(255ng/µL)0.4----H(x10)1-8.610
I732095 8/22 miniprep (1)(430ng/µL)4.61-1-H(x10)3-20.430

9/5

Gel Extraction

Honda and Yamauchi
LaneSample
11kb ladder
29/4 I732095(E,S)-1
3-
49/4 I732095(E,S)-1
5-
69/4 I732095(E,S)-1 control

Genome Cloning

Honda

(1)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer AT cloning primer 61/(µL) Rv Primer AT cloning primer 61/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
50 3 3 4 40 100

(2)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer SAMmt cloning primer 58/(µL) Rv Primer SAMmt cloning primer 57/(µL) DNA CCAP 1023/1/(µL) MilliQ/(µL) Total/(µL)
50 3 3 4 40 100

(3)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer DiDECARB cloning primer 57/(µL) Rv Primer DiDECARB cloning primer 57/(µL) DNA CCAP 1023/1/(µL) MilliQ/(µL) Total/(µL)
50 3 3 4 40 100
Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 55-65°C 72°C 72°C 12°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda

(1)AT

LaneSample
1-
2-
3-
41kb ladder
5(1)-1
6(1)-2
7(1)-3
8(1)-4
9(1)-5
10(1)-6
11(1)-7
12(1)-8
131kb ladder
14-
15-
16-
17-

(2)SAMmt

LaneSample
1-
2-
3-
41kb ladder
5(2)-1
6(2)-2
7(2)-3
8(2)-4
9(2)-5
10(2)-6
11-
12-
13-
14-
15-
16(2)-8
171kb ladder

(3)DiDECARB

LaneSample
1-
2-
3-
41kb ladder
5(3)-1
6(3)-2
7(3)-3
8(3)-4
9(3)-5
10(3)-6
11(3)-7
12(3)-8
131kb ladder
14-
15-
16-
17-

Gel Extraction

Murata
LaneSample
11kb ladder
29/4 I732045(E,S)
39/4 I732045(E,S)
4-
5-
69/4 I732045(E,S)
79/4 I732045(E,S)
8-
91kb ladder
109/4 control

9/8

Genome Cloning and PCR

Honda and Yamaura

(1)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer AT cloning primer 61/(µL) Rv Primer AT cloning primer 61/(µL) 9/5 AT (genome cloning product) (1)-3 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(2)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer DiDECARB cloning primer 61/(µL) Rv Primer DiDECARB cloning primer 61/(µL) 9/5 AT (genome cloning product) (3)-3 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(3)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer Pre-dddD cloning primer/(µL) Rv Primer suf-dddD cloning primer/(µL) 3/19 dddD (DNA purification product)(121µg/µL)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda
LaneSample
11kb ladder
2PCR product (AT)
3PCR product (DIDECARB)
4PCR product (dddD)
5PCR product (dddD)
61kb ladder

DNA Purification

Matsumoto
SampleConcentration/(µg/mL)260/280260/230
9/8 AT551.371.14
9/8 DiDECARB951.693.04
9/8 dddD1451.521.78

Restriction Enzyme Digestion

Matsumoto
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/8 dddD (DNA purificaion and PCR product)13.81-1-H3-11.230
control0.7----H1-8.310

Genome Cloning and PCR

Yasuda
2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer SAMmt cloning primer 58/(µL) Rv Primer SAMmt cloning primer 57/(µL) 9/5 SAMmt (genome cloning product) No.3/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 63°C 72°C 72°C 12°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

9/9

DNA purification

Honda
SampleConcentration/(µg/mL)260/280260/230
9/8 dddD(E,S)1.3(x50)1.661.28

Ligation

Li

(1)Sample

(2)Control

Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
pSB1C34.2dddD3.8(2x)Buffer9T4 Ligase1-18
pSB1C34.2--(2x)Buffer9T4 Ligase13.818

Transformation

Li
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/9 dddD+pSB1C3 ligation product5DH5α5055SOC
9/9 dddD+pSB1C3 ligation product control3DH5α3033SOC

Liquid Culture

Honda
SampleMedium
9/8 T7-RBSPlusgrow

PCR Cloning

Murata
2x KAPA HiFi HotStart ReadyMix/(µL) Fw REDOX cloning primer 59/(µL) Rv REDOX primer Ver.2 64/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
　　　　　　　　　　　　　　　　　　　　 　　　　　　　　　　　　　　　　　　　　 　　　　　　　　　　　　　　　　　　　　
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 64°C 72°C72°C12°C
5min 20sec 15sec 15sec 5min Infinity

Denature-Annealing-Extension 25cycle

Gel Extraction

Murata
LaneSample
11kb ladder
29/8 SAMmt PCR product (5µL)
3-
49/8 SAMmt PCR product (10µL)
59/8 SAMmt PCR product (10µL)
6-

PCR

Honda
2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer SAMmt cloning primer/(µL) Rv Primer SAMmt cloning primer/(µL) 9/9 AT (Gel Extraction product)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 63°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

9/10

Electrophoresis

Murata
LaneSample
1-
2-
31kb ladder (5µL)
49/9 REDOX PCR product (2µL)
59/9 SAMmt PCR product (2µL)
61kb ladder (5µL)
7-
8-

Miniprep

Murata
SampleConcentration/(µg/mL)260/280260/230
T7-RBS2901.901.83

Cloning PCR

Murata
2x KAPA HiFi HotStart ReadyMix/(µL) Fw REDOX cloning primer 59/(µL) Rv REDOX primer Ver.2 64/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

Electrophoresis

Murata
LaneSample
1-
2-
3-
41kb ladder
5SAMmt (x50) (10µL)
6-
7-
8-

Colony PCR

Honda

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
40 1.6 1.6 36.8 80
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Number Sample
1 pSB1C3-dddD(1)
2 pSB1C3-dddD(2)
3 pSB1C3-dddD(3)
4 pSB1C3-dddD(4)
5 pSB1C3-dddD(5)
6 pSB1C3-dddD(6)
7 pSB1C3-dddD (control)
8 control
LaneSample
1-
2-
3-
41kb ladder
59/9 dddD+pSB1C3 (1)
69/9 dddD+pSB1C3 (2)
79/9 dddD+pSB1C3 (3)
89/9 dddD+pSB1C3 (4)
91kb ladder
109/9 dddD+pSB1C3 (5)
119/9 dddD+pSB1C3 (6)
129/9 dddD+pSB1C3 (control)
13control
141kb ladder
15-
16-
17-

Electrophoresis

Yasuda
LaneSample
1-
2-
31kb ladder
4REDOX PCR product
5-

Overlap Extension(OE)-PCR

Murata
10x Buffer for KOD-Plus-Ver.2 2mM dNTPs 25mM MgSO4 Primer(10µM each) #1 Template DNA #2 KOD-Plus-(1U/µL) MilliQ Total
5µL 5µL 3µL 1.5µL 1µL 1µL 33µL 50µL

(1)
#1: AT OE-A, AT OE-B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(2)
#1: AT OE-C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(3)
#1: AT OE-2C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(4)
#1: SAMmt OE-A, SAMmt OE-B
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(5)
#1: SAMmt OE-C, SAMmt OE-D
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(6)
#1: DECARB OE-A, DECARB OE-B<
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(7)
#1: DECARB OE-C, DECARB OE-D
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(8)
#1: DiDECARB OE-A, DiDECARB OE-B
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(9)
#1: DiDECARB OE-C, DiDECARB OE-D
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(10)
#1: AT OE-C, AT OE-2B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)

Genome Cloning

Yasuda
2x KAPA HiFi HotStart ReadyMix/(µL) Fw REDOX cloning primer 59/(µL) Rv REDOX primer Ver.2 64/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
50 3 3 4 40 100
NumberAnnealingTemperature
165.0 °C
264.3 °C
363.0 °C
461.1 °C
558.8 °C
656.9 °C
755.7 °C
855.0 °C

Gel Extraction

Yasuda

(1)

LaneSample
11kb ladder
29/10 OE-PCR(1)
39/10 OE-PCR(1)
4-
59/10 OE-PCR(2)
6-
79/10 OE-PCR(2)
8-
91kb ladder
109/10 OE-PCR(3)
119/10 OE-PCR(3)

(2)

LaneSample
11kb ladder
29/10 OE-PCR(4)
39/10 OE-PCR(4)
4-
59/10 OE-PCR(5)
69/10 OE-PCR(5)
71kb ladder

(3)

LaneSample
1-
21kb ladder
39/10 OE-PCR(6)
49/10 OE-PCR(6)

(4)

LaneSample
1-
21kb ladder
39/10 OE-PCR(7)
49/10 OE-PCR(7)

(5)

LaneSample
1-
21kb ladder
39/10 OE-PCR(8)
49/10 OE-PCR(8)

(6)

LaneSample
11kb ladder
2-
39/10 OE-PCR(9)
49/10 OE-PCR(9)
59/10 OE-PCR(10)
69/10 OE-PCR(10)

Ligation

Tatsui and Murata
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
YC plac111 (SmaI cut)19/8 dddD(DNA purification product)0.6(2x)Buffer5T4 Ligase12.410

Liquid Culture

Honda
SampleMedium
9/9 dddD+pSB1C3-(1)CP

Restriction Enzyme Digestion

Shimizu
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/9 T7-RBS6.9--11H3-18.130
control1----H1-810

OE-PCR

Shimizu
10x Buffer for KOD-Plus-Ver.2 2mM dNTPs 25mM MgSO4 Primer(10µM each) #1 Template DNA #2 KOD-Plus-(1U/µL) MilliQ Total
5µL 5µL 3µL 2µL 0.5µL 1µL 31µL 50µL

(1)
#1: SAMmt OE-A, SAMmt OE-D
#2: 9/10 SAMmt(4)(5) (Gel Extraction Product)
(2)
#1: DECARB OE-A, DECARB OE-D
#2: 9/10 DECARB(6)(7) (Gel Extraction Product)
(3)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/10 DiDECARB(8)(9) (Gel Extraction Product)
(3')
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/10 DiDECARB(3)(9) (Gel Extraction Product)

Predenature Denature Annealing Extension Last Extension Cooling
98°C 98°C 55°C 72°C 72°C 12°C
5min 20sec 15sec 2min10sec 5min Infinity

Denature-Annealing-Extension 25cycle

Ligation

Shimizu
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
YCplac1111dddD1(2x)Buffer5T4 Ligase1210

9/11

Miniprep

Murata
SampleConcentration/(µg/mL)260/280260/230
9/10 dddD (liquid culture product)1651.882.83

DNA Purification

Matsumoto
SampleConcentration/(µg/mL)260/280260/230
9/10 REDOX(5)(6) (genome cloning product)551.561.32

OE-PCR

Shimizu
10x Buffer for KOD-Plus-Ver.2 2mM dNTPs 25mM MgSO4 Primer(10µM each) #1 Template DNA #2 KOD-Plus-(1U/µL) MilliQ Total
5µL 5µL 3µL 1.5µL 1µL 1µL 32µL 50µL

(1)
#1: AT OE-A, AT OE-B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(2)
#1: AT OE-C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(3)
#1: AT OE-2C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(4)
#1: REDOX (Forward with prefix), REDOX (Reverse with sufix) #2: 9/11 REDOX (DNA Purification Product)(55µg/mL)
(5)(6)
#1: SAMmt OE-A, SAMmt OE-B
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(7)(8)
#1: SAMmt OE-C, SAMmt OE-D
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(9)(10)
#1: DECARB OE-A, DECARB OE-B<
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(11)(12)
#1: DECARB OE-C, DECARB OE-D
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(13)(14)
#1: DiDECARB OE-A, DiDECARB OE-B
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(15)(16)
#1: DiDECARB OE-C, DiDECARB OE-D
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)

Electrophoresis

Honda and Murata
LaneSample
1-
21kb ladder
39/10 OE-PCR-(1)
49/10 OE-PCR-(2)
59/10 OE-PCR-(3)
69/10 OE-PCR-(3')
71kb ladder

Colony PCR

Murata and Yamauchi

Master Mix (x32)

Quick Taq/(µL) M13 Fw/(µL) M13 Rv/(µL) MilliQ/(µL) Total/(µL)
160 6.4 6.4 147.2 320
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 72sec

Denature-Annealing-Extension 30cycle

(1)

Number Sample
1LB Amp. 9/10 mamB(1)
2LB Amp. 9/10 mamB(2)
3LB Amp. 9/10 mamB(3)
4LB Amp. 9/10 mamB(4)
5LB Amp. 9/10 mamB(5)
6LB Amp. 9/10 mamB(6)
7LB Amp. 9/10 mamB(7)
8LB Amp. 9/10 mamB(8)

(2)

Number Sample
1LB Amp. 9/10 mamL(1)
2LB Amp. 9/10 mamL(2)
3LB Amp. 9/10 mamL(3)
4LB Amp. 9/10 mamL(4)
5LB Amp. 9/10 mamL(5)
6LB Amp. 9/10 mamL(6)
7LB Amp. 9/10 mamL(7)
8LB Amp. 9/10 mamL(8)

(3)

Number Sample
1LB Amp. 9/10 mamQ(1)
2LB Amp. 9/10 mamQ(2)
3LB Amp. 9/10 mamQ(3)
4LB Amp. 9/10 mamQ(4)
5LB Amp. 9/10 mamQ(5)
6LB Amp. 9/10 mamQ(6)
7LB Amp. 9/10 mamQ(7)
8LB Amp. 9/10 mamQ(8)

(4)

Number Sample
1LB Amp. 9/10 dddD(1)
2LB Amp. 9/10 dddD(2)
3LB Amp. 9/10 dddD(3)
4LB Amp. 9/10 dddD(4)
5LB Amp. 9/10 dddD(5)
6LB Amp. 9/10 dddD(6)
7LB Amp. 9/10 dddD(7)
8LB Amp. 9/10 dddD(8)

Mutation PCR

Honda

(1)dddD (PstI)

Prime STAR MAX Premix(2x) Fw dddD PstI mutation primer Rv dddD PstI mutation primer 9/11 MP dddD (x2000) MilliQ Total
25µL 1µL 1µL 1µL 22µL 50µL

(2)T7-RBS-Histag

Prime STAR MAX Premix(2x) Fw pSB1C3-his-F-v2 primer Rv pSB1C3 Histag mutation primer 9/10 MP T7-RBS (x3000) MilliQ Total
25µL 1µL 1µL 1µL 22µL 50µL

(3)Control

Prime STAR MAX Premix(2x) Fw Control primer Rv Control primer Control template (x1000) MilliQ Total
25µL 0.5µL 0.5µL 1µL 23µL 50µL
Predenature Denature Annealing Extension Cooling
98°C 98°C 55°C 72°C 12°C
5min 10sec 15sec 25sec Infinity

Denature-Annealing-Extension 30cycle

DNA Purification

Honda
SampleConcentration/(µg/mL)260/280260/230
dddD, T7-RBS-His---

Gel Extraction

Honda

(1)

LaneSample
11kb ladder
29/11 OE-PCR-(1)
39/11 OE-PCR-(1)
4-
5-
69/11 OE-PCR-(2)
79/11 OE-PCR-(2)
8-
91kb ladder
109/11 OE-PCR-(3)
119/11 OE-PCR-(3)

(2)

LaneSample
11kb ladder
29/11 OE-PCR-(6)
39/11 OE-PCR-(6)
4-
5-
69/11 OE-PCR-(8)
79/11 OE-PCR-(8)
8-
91kb ladder
109/11 OE-PCR-(10)
119/11 OE-PCR-(10)

(3)

LaneSample
11kb ladder
29/11 OE-PCR-(12)
39/11 OE-PCR-(12)
49/11 OE-PCR (REDOX 2nd PCR)
5-
69/11 OE-PCR-(14)
79/11 OE-PCR-(14)
8-
91kb ladder
109/11 OE-PCR-(16)
119/11 OE-PCR-(16)

Gel Extraction

Li
Sample Concentration/(µg/mL) 260/280 260/230
(1) 0.1 - -
(2) 4.9 1.67 1.95
(3) 4.6 1.74 1.24
(6) 6.3 1.61 2.03
(8) 2.6 1.19 0.95
(10) 8.5 1.52 1.37
(12) 1.8 1.44 1.51
(14) 2.4 1.85 0.82
(16) 5.0 1.72 2.21

DNA Purification

Yamauchi
SampleConcentration/(µg/mL)260/280260/230
9/10 OE-PCR (1) SAMmt9751.061.08
9/10 OE-PCR (2) DECARB4201.672.19
9/20 OE-PCR (3) DiDECARB1751.662.45
9/10 OE-PCR (3') DiDECARB2151.571.82
9/11 Mutation PCR dddD2751.611.92
9/11 Mutation PCR T71401.491.78
Control C3401.621.47

Ligation

Tatsui and Yamauchi
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
YC plac111 (SmaI cut)19/11 SAMmt (DNA purification product)0.5(2x) Buufer5T4 Ligase12.59
YC plac111 (SmaI cut)19/11 DiDECARB (DNA purification product)1(2x) Buufer5T4 Ligase129
YC plac111 (SmaI cut)19/11 DECARB (DNA purification product)1(2x) Buufer5T4 Ligase129
YC plac111 (SmaI cut)19/11 REDOX (DNA purification product)2(2x) Buufer5T4 Ligase119
9/10 Pcon-X(PstI cut, gel extraction product)1.5mamL(X.P)2(2x)Buffer5T4 Ligase1-9.5
9/10 Pcon-X(PstI cut, gel extraction product)1.5mamQ(X.P)2(2x)Buffer5T4 Ligase1-9.5

OE-PCR

Yasuda
10x Buffer for KOD-Plus-Ver.2 2mM dNTPs 25mM MgSO4 Primer(10µM each) #1 Template DNA #2 KOD-Plus-(1U/µL) MilliQ Total
5µL 5µL 3µL 2µL 0.5µL 1µL 31µL 50µL

(1)
#1: AT OE-A, AT OE-D
#2: 9/10 OE-PCR(10), 9/11 OE-PCR(1), 9/11 OE-PCR(3)
(2)
#1: SAMmt OE-A, SAMmt OE-D
#2: 9/11 OE-PCR(6), 9/11 OE-PCR(8)
(3)
#1: DECARB OE-A, DECARB OE-D
#2: 9/11 OE-PCR(10), 9/11 OE-PCR(12)
(4)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/11 OE-PCR(14), 9/11 OE-PCR(16)

Predenature Denature Annealing Extension Last Extension Cooling
98°C 98°C 55°C 72°C 72°C 12°C
2min 10sec 10sec 1min 5min Infinity

Denature-Annealing-Extension 25cycle

9/12

Electrophoresis

Li
LaneSample
1Blank
21kb ladder
39/11 OE-PCR 2nd (1)
49/11 OE-PCR 2nd (2)
59/11 OE-PCR 2nd (3)
69/11 OE-PCR 2nd (4)

DNA Purification

Li
SampleConcentration/(µg/mL)260/280260/230
9/11 OE-PCR 2nd (1)2201.651.96
9/11 OE-PCR 2nd (2)2401.652.10
9/11 OE-PCR 2nd (3)2251.682.03
9/11 OE-PCR 2nd (4)1601.641.91

Transformation

Yamauchi
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/11 SAMmt (ligation product)2DH5α1012CP
9/11 DiDECARB (ligation product)2DH5α1012CP
9/11 DECARB (ligation product)2DH5α1012CP
9/11 REDOX (ligation product)2DH5α1012CP

Sequence

Yasuda
Bis Dye v3.1 Sequencing RR-24(/µL) Bis Dye 5x Sequencing Buffer(/µL) Primer (10µM) #1(/µL) 9/11 dddD miniprep (165ng/nL)(/µL) MilliQ Total
4 2 3.2 1.5 9.3 20

1. VF
2. Sequence No.1
3. Sequence No.2
4. Sequence No.3
5. Sequence No.4
6. Sequence No.5
7. Sequence No.6

Liquid Culture

Honda
SampleMedium
9/10 dddD-YCplac111 (Transformation product)Amp

Restriction Enzyme Digestion

Murata
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/10 OE-PCR SAMmt21--1H3-2330
9/10 OE-PCR DECARB4.8-11-M3317.230
9/10 OE-PCR DiDECARB12.4-11-M3310.630
9/10 OE-PCR T7-RBS6.9--11H3-18.130
Control2.3----H1-59.9

Genome Cloning PCR

Honda

(1)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer SAMmt cloning primer/(µL) Rv Primer SAMmt cloning primer/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(2)

2K KAPA HiFi HotStart ReadyMix/(µL) Fw Primer DECARB cloning primer/(µL) Rv Primer DECARB cloning primer/(µL) DNA CCAP 1023/1/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

Gel Extraction

Honda
LaneSample
11kb ladder
2Control(9/11 T7-RBS)
3-
49//11 T7-RBS(S,P)
5-
61kb ladder

Cloning PCR (for Histag)

Murata
2x KAPA HiFi HotStart ReadyMix/(µL) Fw 9/10 AT OE-A/(µL) Rv 9/12 AT His/(µL) Templete 9/11 OE-PCR 2nd (1) 9/12/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 pick up 11 25
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 65°C 72°C 72°C 12°C
5min 20sec 15sec 45sec 5min Infinity

Denature-Annealing-Extension 25cycle

Miniprep

Tatsui
SampleConcentration/(µg/mL)260/280260/230
dddD-pSB1C3 (Liquid culture product)3701.752.48

DNA Purification

Tatsui
SampleConcentration/(µg/mL)260/280260/230
9/11 DECARB (X,S)---
9/11 DiDECARB (X,S)---
9/11 SAMmt (X,S)---

Restriction Enzyme Digestion

Tatsui
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/12 AT (DNA purification product)91--1H3-1630

PCR (for Control)

Murata

(1)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw AT-A/(µL) Rv AT-D/(µL) Templete 9/8 AT (DNA purification product)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(2)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw SAMmt-A/(µL) Rv SAMmt-D/(µL) Templete 9/12 AT (Genome cloning product)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(3)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw DECARB-A/(µL) Rv DECARB-D/(µL) Templete 9/12 DECARB (genome cloning product)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25

(4)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw DiDECARB-A/(µL) Rv DiDECARB-D/(µL) Templete 9/8 AT (DNA purification product)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 1 10 25
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min Infinity

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yamauchi
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/12 AT-His (DNA purification product)8.7-1-1M3313.330
Control0.4----M306.610
8/2 Pcon201-1-H33230
Controlpick up----H30710

Ligation

Honda
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
YC plac1111AT-His cloning0.5(2x)Buffer5T4 Ligase13.510
YC plac1111AT0.5(2x)Buffer5T4 Ligase13.510
pSB1C3 (E,P)1.5SAMmt (E,P)1(2x)Buffer5T4 Ligase12.510

DNA Purification

Tatsui
SampleConcentration/(µg/mL)260/280260/230
9/11 DECARB (Restriction Enzyme Digestion product)401.741.95
9/11 DiDECARB (Restriction Enzyme Digestion product)302.001.70
9/11 SAMmt (Restriction Enzyme Digestion product)203.542.33
9/11 T7-RBS601.810.27
9/10 T7-RBS501.651.77

Electrophoresis

LaneSample
1-
2-
3-
41kb ladder
59/11 E-PCR 2nd (1)
69/11 Control (PCR product) (1)
79/11 E-PCR 2nd (2)
89/11 Control (PCR product) (2)
99/11 E-PCR 2nd (3)
109/11 Control (PCR product) (3)
119/11 E-PCR 2nd (4)
129/11 Control (PCR product) (4)
131kb ladder
14-
15-
16-

Transformation

Honda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
AT-His-YCplac1111DH5α1011SOD
AT-YCplac1111DH5α1011SOD
SAMmt(E,P)-pSB1C3(E,P)1DH5α1011SOD

Transformation

Yasuda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/11 dddD (DNA purification product)1DH5α1011SOD
9/11 T7-His (DNA purification product)1DH5α1011SOD

Colony PCR

Tatsui and Li
Predenature Denature Annealing Extension Last Extension Cooling
94°C 94°C 55°C 68°C 68°C 12°C
2min 30sec 30sec 1min 1sec Infinity

Denature-Annealing-Extension 25cycle

LaneSample
1-8YCplac111-SAMmt (1)-(8)
91kb ladder
10-16YCplac111-REDOX (1)-(7)

Liquid Culture

Yamauchi
SampleMedium
9/5 Pcon-pSB1C3-(3)CP
9/5 Pcon-pSB1C3-(3)CP
9/11 SAMmt-(3)Amp
9/8 T7-RBS-(1)CP
9/8 T7-RBS-(1)CP

Colony PCR

Honda

Master Mix

Quick Taq/(µL) M13 Fw/(µL) M13 Rv/(µL) MilliQ/(µL) Total/(µL)
35 1.4 1.4 32.2 70
Predenature Denature Annealing Extension Last Extension Cooling
94°C 94°C 55°C 68°C 68°C 12°C
2min 30sec 30sec 1min 1min Infinity

Denature-Annealing-Extension 30cycle

LaneSample
1-8written in "magnetosome"
91kb ladder
10-17REDOX-YCplac111 (1)-(8)

9/13

Miniprep

Honda
SampleConcentration/(µg/mL)260/280260/230
9/12 T7-RBS-pSB1C3 (liquid culture product)952.022.72
9/12 SAMmt-YCplac111 (liquid culture product)1101.701.15

Gel Extraction

Honda and Yasuda
LaneSample
11kb ladder
2-
3-
4pSB1C3-Pcon(X,S)
5-
6Control (pSB1C3-Pcon(X,S))
Sample Concentration/(µg/mL) 260/280 260/230
pSB1C3-Pcon X,S 1.5 1.11 0.59

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
9/12 AT (E,P)2.31.331.11
9/12 AT-His(X,P)2.01.330.86

Ligation

Honda
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
9/8 pSB1C3(E,P)39/13 AT(E,P)(DNA purification product)1(2x)Buffer5T4 Ligase1010
9/13 pSB1C3(X,P)39/13 AT-His(X,P)(DNA purification product)1(2x)Buffer5T4 Ligase1010

Miniprep

Honda
SampleConcentration/(µg/mL)260/280260/230
9/12 SAMmt-YCplac1111401.801.90
T7-RBS-pSB1C32901.781.65

Liquid Culture

Yasuda
SampleMedium
9/12 dddD-pSB1C3-(1)CP
9/12 dddD-pSB1C3-(2)CP
9/12 dddD-pSB1C3-(3)CP
9/12 T7-RBS-His-(1)CP
9/12 T7-RBS-His-(2)CP
9/12 T7-RBS-His-(3)CP
Control

9/14

Colony PCR

Yasuda
Quick Taq/(µL) M13 Fw/(µL) M13 Rv/(µL) MilliQ/(µL) Total/(µL)
335 13.4 13.4 368.2 670
Predenature Denature Annealing Extension Cooling
94°C 94°C 55°C 68°C 12°C
2min 30sec 30sec 160sec infinity

Denature-Annealing-Extension 30cycle

1~8: 9/12 AT-YCplac111(1)~(8)
9~16: 9/12 AT His-YCplac111(1)~(8)
17~24: 9/12 mamB-YCplac111(1)~(8)
25~44: 9/12 DECARB-YCplac111(1)~(20)
45~64: 9/12 DiDECARB-YCplac111(1)~(20)

Gel Extraction

Honda
LaneSample
11kb ladder
29/13 Control (SAMmt)
39/13 SAMmt (E,P)
4-
59/13 Control (Pcon-pSB1C3)
69/13 Pcon-pSB1C3(X,S)

DNA Purification

Honda
SampleConcentration/(µg/mL)260/280260/230
Pcon-pSB1C3(X,S)400.131.27

Electrophoresis

Yasuda

(1)

LaneSample
19/12 AT-YCplac111(1)
29/12 AT-YCplac111(2)
39/12 AT-YCplac111(3)
49/12 AT-YCplac111(4)
59/12 AT-YCplac111(5)
69/12 AT-YCplac111(6)
79/12 AT-YCplac111(7)
89/12 AT-YCplac111(8)
91kbp GereRuler
109/12 AT His-YCplac111(1)
119/12 AT His-YCplac111(2)
129/12 AT His-YCplac111(3)
139/12 AT His-YCplac111(4)
149/12 AT His-YCplac111(5)
159/12 AT His-YCplac111(6)
169/12 AT His-YCplac111(7)
179/12 AT His-YCplac111(8)

(2)

LaneSample
19/12 mamB-YCplac111(1)
29/12 mamB-YCplac111(2)
39/12 mamB-YCplac111(3)
49/12 mamB-YCplac111(4)
59/12 mamB-YCplac111(5)
69/12 mamB-YCplac111(6)
79/12 mamB-YCplac111(7)
89/12 mamB-YCplac111(8)
91kbp GereRuler
109/12 DECARB-YCplac111(1)
119/12 DECARB-YCplac111(2)
129/12 DECARB-YCplac111(3)
139/12 DECARB-YCplac111(4)
149/12 DECARB-YCplac111(5)
159/12 DECARB-YCplac111(6)
169/12 DECARB-YCplac111(7)
179/12 DECARB-YCplac111(8)

(3)

LaneSample
19/12 DECARB-YCplac111(9)
29/12 DECARB-YCplac111(10)
39/12 DECARB-YCplac111(11)
49/12 DECARB-YCplac111(12)
59/12 DECARB-YCplac111(13)
69/12 DECARB-YCplac111(14)
79/12 DECARB-YCplac111(15)
89/12 DECARB-YCplac111(16)
91kbp GereRuler
109/12 DECARB-YCplac111(17)
119/12 DECARB-YCplac111(18)
129/12 DECARB-YCplac111(19)
139/12 DECARB-YCplac111(20)
149/12 DiDECARB-YCplac111(1)
159/12 DiDECARB-YCplac111(2)
169/12 DiDECARB-YCplac111(3)
179/12 DiDECARB-YCplac111(4)

(4)

LaneSample
19/12 DiDECARB-YCplac111(5)
29/12 DiDECARB-YCplac111(6)
39/12 DiDECARB-YCplac111(7)
49/12 DiDECARB-YCplac111(8)
59/12 DiDECARB-YCplac111(9)
69/12 DiDECARB-YCplac111(10)
79/12 DiDECARB-YCplac111(11)
89/12 DiDECARB-YCplac111(12)
91kbp GereRuler
109/12 DiDECARB-YCplac111(13)
119/12 DiDECARB-YCplac111(14)
129/12 DiDECARB-YCplac111(15)
139/12 DiDECARB-YCplac111(16)
149/12 DiDECARB-YCplac111(17)
159/12 DiDECARB-YCplac111(18)
169/12 DiDECARB-YCplac111(19)
179/12 DiDECARB-YCplac111(20)

Miniprep

Honda
SampleConcentration/(µg/mL)260/280260/230
9/13 dddD-pSB1C3(1)6451.821.70
9/13 dddD-pSB1C3(2)6601,691.74
9/13 dddD-pSB1C3(3)6351.871.57
9/13 T7-RBS-His-pSB1C3(1)4401.651.23
9/13 T7-RBS-His-pSB1C3(2)4501.921.46
9/13 T7-RBS-His-pSB1C3(3)1901.441.26

Restriction Enzyme Digestion

Honda
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/14 dddD-pSB1C3(1)6-2-2M663860
9/14 dddD-pSB1C3(2)6-2-2M663860
9/14 dddD-pSB1C3(3)6-2-2M663860
9/14 T7-RBS-His-pSB1C3(1)5--11H3-1830
9/14 T7-RBS-His-pSB1C3(2)5--11H3-1830
9/14 T7-RBS-His-pSB1C3(3)10--11H3-1530

Dephosphorylation

Honda
Sample/(µL)CIAP/(µL)
9/14 pSB1C3(X,S)(gel extraction product)200.25
SampleConcentration/(µg/mL)260/280260/230
9/14 pSB1C3(X,S)(gel extraction product)602.930.16

Ligation

Yasuda
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
Pcon-pSB1C3(X,S)(Dephosphorylation product)0.839/12 DECARB(X,S)(DNA purification product)2.9310x Lig Buffer2NEB T4 DNA Ligase113.2420
Pcon-pSB1C3(X,S)(Dephosphorylation product)0.839/12 DECARB(X,S)(DNA purification product)3.6610x Lig Buffer2NEB T4 DNA Ligase112.5120

9/15

Colony PCR

Tatsui

Master Mix(x24)

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
120 4.8 4.8 110.4 240
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

Denature-Annealing-Extension 30cycle

Number Sample
1~8 9/14 AT-pSB1C3

Electrophoresis

Tatsui
LaneSample
11kb ladder
2~99/14 AT-pSB1C3

Gel Extraction

Tatsui
LaneSample
11kb ladder
2-
39/14 dddD-pSB1C3(X,P)(After mutation)-(1)
4-
51kb ladder
6-
79/14 dddD-pSB1C3(X,P)(After mutation)-(2)
8-
91kb ladder
10-
119/14 dddD-pSB1C3(X,P)(After mutation)-(3)

DNA Purification

Tatsui
SampleConcentration/(µg/mL)260/280260/230
9/14 dddD-pSB1C3(X,P)(After mutation)-(1)1.7 (x50)1.370.33
9/14 dddD-pSB1C3(X,P)(After mutation)-(2)1.6 (x50)1.410.36
9/14 dddD-pSB1C3(X,P)(After mutation)-(3)2.2 (x50)1.230.42
T7-RBS-pSB1C3(S cut)2.0 (x50)2.110.34

Restriction Enzyme Digestion

Murata
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/13 T7-RBS-pSB1C37--11H3-930
9/11 REDOX (DNA purification product)101--1H3-1530
9/13 Pcon-pSB1C3251--1H3-030
9/15 T7-RBS-pSB1C325---1M3-130

PCR Cloning

Murata
2x KAPA HiFi Hotstart Ready Mix/(µL) 9/10 Suf-REDOX Rv/(µL) 9/10 Fw Re-REDOX 59/(µL) 9/11 REDOX 5.6(DNA purification product) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 0.2 11 25.2
Predenature Denature Annealing Extension
95°C 98°C 65°C 72°C
5min 20sec 15sec 50sec

Denature-Annealing-Extension 25cycle

Gel Extraction

Murata
LaneSample
11kb ladder
2-
39/14 T7-RBS-His-pSB1C3(S,P)-(1)
4-
51kb ladder
6-
79/14 T7-RBS-His-pSB1C3(S,P)-(1)
8-
91kb ladder
10-
119/14 T7-RBS-His-pSB1C3(S,P)-(1)

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
9/15 REDOX (PCR product)4.91.760.66

Ligation

Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
YCplac111 (SmaI cut)(x3)19/12 AT His (DNA purification product)0.3(x2)Buffer5T4 Ligase12.710
YCplac111 (SmaI cut)(x3)19/15 REDOX (DNA purification product)0.2(x2)Buffer5T4 Ligase12.810
YCplac111 (SmaI cut)(x3)19/12 DECARB(DNA purification product)0.4(x2)Buffer5T4 Ligase12.610
YCplac111 (SmaI cut)(x3)19/12 DiDECARB(DNA purification product)0.3(x2)Buffer5T4 Ligase12.710

Liquid Culture

Honda
SampleMedium
9/14 AT-pSB1C3 (Transformation product)-(1)SOD
9/14 AT-pSB1C3 (Transformation product)-(2)SOD
9/14 AT-pSB1C3 (Transformation product)-(3)SOD

Dephosphorylation

Honda
Sample/(µL)CIAP/(µL)
9/15 T7-RBS-pSB1C3(P)(Restriction Enzyme Digestion product)200.25
9/13 pSB1C3(E,P)(Gel extraction product)200.25
37°C 50°C 75°C
30min 1h 15min

Gel Extraction

Yasuda
LaneSample
11kb ladder
2-
39/14 T7-RBS-His-pSB1C3(S,P)-(1)
4-
5-
6-
79/14 T7-RBS-His-pSB1C3(S,P)-(2)
8-
9-
10-
119/14 T7-RBS-His-pSB1C3(S,P)-(3)
SampleConcentration/(µg/mL)260/280260/230
9/14 T7-RBS-His-pSB1C3(S,P)-(1)251.810.50
9/14 T7-RBS-His-pSB1C3(S,P)-(2)251.130.21
9/14 T7-RBS-His-pSB1C3(S,P)-(3)251.650.09

Restriction Enzyme Digestion

Yasuda
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/14 T7-RBS-His-pSB1C3 (Miniprep product)13.63--33H9-61.3790
Control0.3----H1-8.910

Transformation

Murata
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
AT-His-YCplac11110DH5α212CP
REDOX-YCplac11110DH5α212CP
DECARB-pSB1C310DH5α212CP
DiDECARB-pSB1C310DH5α212CP

Ligation

Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
9/15 T7-RBS(S,P)(Dephosphorylation product)2.09/15 dddD(After mutation)(X,P)(Gel extraction product)2.0(x2)Buffer5T4 Ligase1010
9/15 pSB1C3 (E,P)(Dephosphorylation product)0.49/12 SAMmt (E,P)(DNA purification product)2.9(x2)Buffer5T4 Ligase10.710
9/15 pSB1C3 (E,P)(Dephosphorylation product)1.39/15 REDOX (E,P)(DNA purification product)2.4(x2)Buffer5T4 Ligase10.310
9/15 T7-RBS(S,P)(Gel extraction product)2.69/15 AT His (X,P)(DNA purification product)1.2(x2)Buffer5T4 Ligase10.210

Transformation

Murata and Yasuda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
DECARB-YCplac1112DH5α1012SOD
DiDECARB-YCplac1112DH5α1012SOD
dddD(After mutation)-T7-RBS-pSB1C32DH5α1012SOD
SAMmt-pSB1C32DH5α1012SOD
REDOX-pSB1C32DH5α1012SOD
AT-T7-RBS-pSB1C32DH5α1012SOD

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
9/15 REDOX (E,P)45--

9/16

Miniprep

Yasuda
SampleConcentration/(µg/mL)260/280260/230
9/15 AT-pSB1C3-(1)2801.812.08
9/15 AT-pSB1C3-(1)2051.641.32
9/15 AT-pSB1C3-(1)3201.661.61

PCR (for Sequence)

Tatsui and Nakamura

9/14 dddD-pSB1C3(Miniprep product)-(1)
(1)dddD sequence VF2

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(2)dddD sequence No.1

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(3)dddD sequence No.2

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(4)dddD sequence No.3

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(5)dddD sequence No.4

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(6)dddD sequence No.5

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(7)dddD sequence No.6

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5
Predenature Denature Annealing Extension Final Extension Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min Infinity

Denature-Annealing-Extension 30cycle

9/14 dddD-pSB1C3(Miniprep product)-(2)

(1)dddD sequence VF2

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(2)dddD sequence No.1

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(3)dddD sequence No.2

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(4)dddD sequence No.3

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(5)dddD sequence No.4

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(6)dddD sequence No.5

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

(7)dddD sequence No.6

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
0.63 0.5 1.75 0.5 6.6 10.5

9/15 AT-pSB1C3(Miniprep product)-(1)

(1)VF

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
1.43 0.5 1.75 0.5 5.8 10.5

(2)AT for Sequence

Templete DNA/(µL) Big Dye/(µL) (x5)Buffer/(µL) Primer MilliQ/(µL) Total/(µL)
1.43 0.5 1.75 0.5 5.8 10.5
Predenature Denature Annealing Extension Final Extension Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min Infinity

Denature-Annealing-Extension 30cycle

Dephosphorylation

Honda
Sample/(µL)
1.*(written in magnetosome)*
2.**
3.9/15 T7-RBS-pSB1C3(P)30
4.9/15 T7-RBS-pSB1C3(S,P)30
5.9/15 T7-RBS-pSB1C3(S,P)30
6.9/15 T7-RBS-pSB1C3(S,P)30
7.9/15 T7-RBS-pSB1C3(S,P)30

Colony PCR

Yasuda

Master Mix

Quick Taq/(µL) M13 Ew/(µL) M13 Rv/(µL) MilliQ/(µL) Total/(µL)
300 12 12 276 600

Electrophoresis

Yasuda

(1)

LaneSample
1-89/15 REDOX-YCplac111
91kb ladder
10-179/15 DECARB-YCplac111

(2)

LaneSample
1-89/15 DiDECARB-YCplac111
91kb ladder
10-179/15 AT-His-YCplac111

(3)

LaneSample
11kb ladder
2-89/15 AT-His-YCplac111

(4)

LaneSample
11kb ladder
2-69/15 AT-His-YCplac111

Gel Extraction

Yamauchi

(1)

LaneSample
11kb ladder
2-
3(written in "magnetosome")
4-
51kb ladder
6-
7(written in "magnetosome")
8-
91kb ladder
10-
119/15 pSB1C3(E,P)

(2)

LaneSample
11kb ladder
2-
39/15 T7-RBS-pSB1C3(P)
4-
51kb ladder
6-
79/15 T7-RBS-pSB1C3(S,P)
8-
91kb ladder
10-
119/15 T7-RBS-pSB1C3(S,P)
SampleConcentration/(µg/mL)260/280260/230
7. 9/15 T7-RBS-pSB1C3(S,P)901.400.44

(3)

LaneSample
1-
21kb ladder
3-
49/15 T7-RBS-pSB1C3(S,P)
5-
61kb ladder
SampleConcentration/(µg/mL)260/280260/230
4. 9/15 T7-RBS-pSB1C3(S,P)801.301.02

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
9/15 T7-RBS-pSB1C3(S,P)(Dephosphorylation product)0.7(x50)1.811.13
9/15 pSB1C3(E,P)(Dephosphorylation product)1.1(x50)1.591.25

Ligation

Yasuda
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
9/16 T7-RBS(S,P)(DNA purification product)1.79/15 dddD(After mutation)(X,P)(gel extraction product)2(x2)Buffer5T4 Ligase10.310
9/16 pSB1C3 (E,P)(DNA purification product)0.559/15 SAMmt (E,P)(gel extraction product)2.9(x2)Buffer5T4 Ligase10.5510
9/16 pSB1C3 (E,P)(DNA purification product)1.69/15 REDOX (E,P)(gel extraction product)2.2(x2)Buffer5T4 Ligase10.210
9/16 T7-RBS-His(S,P)(DNA purification product)2.69/15 AT His(X,P)(gel extraction product)1.2(x2)Buffer5T4 Ligase10.210

Dephosphorylation

Murata
Sample/(µL)CIAPTotal
9/13 pSB1C3 (X,S)(gel extraction product)160.216.2

Transformation

Yasuda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/16 AT-His-T7-RBS-His-DH5α---
9/16 REDOX-pSB1C3-DH5α---
9/16 SAMmt-pSB1C3-DH5α---
9/16 dddD-T7-RBS(After mutation)-DH5α---
9/16 AT-His-YCplac111-DH5α---
9/16 REDOX-YCplac111-DH5α---
9/16 DECARB-YCplac111-DH5α---
9/16 DiDECARB-YCplac111-DH5α---

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
9/16 pSB1C3(X,S)(Dephosphorylation product)351.690.56

Liquid Culture

Yasuda
SampleMedium
9/16 AT RBS His-T7 RBS His(1)-
9/16 AT RBS His-T7 RBS His(2)-

PCR

Yasuda

(1)

9/11 OE-PCR SAMmt 2nd-(2) 2x KAPA HiFi Hotstart Ready Mix/(µL) Fw Primer/(µL) Rv Primer/(µL) MilliQ/(µL) Total/(µL)
pick up 12.5 0.75 0.75 11 25
9/11 OE-PCR DECARB 2nd-(3) 2x KAPA HiFi Hotstart Ready Mix/(µL) Fw Primer/(µL) Rv Primer/(µL) MilliQ/(µL) Total/(µL)
pick up 12.5 0.75 0.75 11 25
Predenature Denature Annealing Extension
95°C 98°C 65°C 72°C
5min 20sec 15sec 50sec

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yasuda
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/11 OE-PCR SAMmt 2nd-(2)8.20.5--0.5H1.504.315
9/15 AT-pSB1C3-(3)(Miniprep product)9.8-1-1M3312.230
Control0.5----M117.510

Ligation

Honda
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
pSB1C31.19/12 DECARB(X,E)(DNA purification product)2.9(x2)Buffer5T4 Ligase1010
pSB1C30.859/12 DiDECARB(X,E)(DNA purification product)2.2(x2)Buffer5T4 Ligase10.9510

Transformation

Honda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/16 DECARB-pSB1C31DH5α1011-
9/16 DiDECARB-pSB1C31DH5α1011-

15min on ice - 1min heat shock(42°C) - 45min preculture (42°C)

9/17

Cycle PCR

Murata and Nakamura

16 Cycle

BigDye Terminator v3.1 Cycle Sequencing RR-24/(µL) BigDye Terminator v3.1 v1.1 5x Sequencing Buffer/(µL) Primer #1/(pmol) Templete DNA #2/(ng) MilliQ /(µL) Total/(µL)
2 1 1.6 75-150 up to 10 10


(1)
#1: VF2
#2: 9/11 dddD (miniprep product) 1µL

#1: Sequence No.5
#2: 9/11 dddD (miniprep product) 1µL


(2)
#1: VF2
#2: 9/14 dddD(After mutation)(miniprep product) 0.25µL

#1: VR
#2: 9/14 dddD(After mutation)(miniprep product) 0.25µL


(3)
#1: VF2
#2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL

#1: Sequence No.5
#2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL


(4)
#1: VR
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL

#1: VF2
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL

#1: Sequence No.1
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL

#1: Secuence No.2
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL

Predenature Denature Annealing Extension Final Extension Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min Infinity

Denature-Annealing-Extension 30cycle

Gel Extraction

Murata
LaneSample
11kb ladder
2Control
3-
49/16 AT-pSB1C3(X,P)
5-
6-

DNA Purification

Murata and Yasuda
SampleConcentration/(µg/mL)
9/12 SAMmt (E,P)75
9/16 SAMmt (PCR product)140
9/16 DECARB (PCR product)230

PCR

Yasuda
10x Buffer for KOD-Plus-Ver.2 2mM dNTPs 25mM MgSO4 Primer(10µM each) #1 Template DNA #2 KOD-Plus-(1U/µL) MilliQ Total
5µL 5µL 3µL 2µL 0.5µL 1µL 31.5µL 50µL


(1)
#1: Pre-REDOX, Suf-REDOX
#2: 9/11 REDOX (DNA purification product)

(2)
#1: Pre-REDOX, Rv-REDOX cloning for His
#2: 9/11 REDOX (DNA purification product)

(3)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/11 DiDECARB (for OE-PCR 2nd)(9/12 DNA purification product)

Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 55°C 72°C 72°C 12°C
2min 10sec 10sec 1min 5min Infinity

Denature-Annealing-Extension 25cycle

DNA Purification

Yasuda
SampleConcentration/(µg/mL)
9/17 REDOX PCR435
9/17 REDOX-His PCR365
9/17 DiDECARB PCR230

Colony PCR

Honda,Nakamura and Matsumoto

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
85 3.4 3.4 78.2 170
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 2min30sec

Denature-Annealing-Extension 30cycle

Number Sample
1-2 9/15 AT-His-T7-RBS-His (1)-(2)
3-7 9/16 AT-His-T7-RBS-His (1)-(5)
8-9 9/16 dddD-T7-RBS (After mutation)(1)-(2)
10-16 9/16 SAMmt-pSB1C3 (1)-(7)

Restriction Enzyme Digestion

Murata
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/17 REDOX-His (DNA purification product)5.5-1-1M3316.530
9/17 REDOX (DNA purification product)4.6-1-1M3317.430

Ligation

Yasuda
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
9/16 T7-RBS(S,P) (DNA purification product)1.49/17 AT(X,P) (DNA purification product)2.3(x2)Buffer5T4 Ligase10.310
YCplac111(SmaI cut)19/17 REDOX-His (DNA purification product)0.6(x2)Buffer5T4 Ligase12.3910
9/16 pSB1C3(E,P) (DNA purification product)0.89/17 SAMmt(E,P) (DNA purification product)1.9(x2)Buffer5T4 Ligase10.810
YCplac111(SmaI cut)1--(x2)Buffer5T4 Ligase1310

Transformation

Yasuda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
9/17 AT-T7-RBS (Ligation product)2DH5α1012SOC
9/17 REDOX-His-YCplac111 (Ligation product)2DH5α1012SOC
9/17 SAMmt-pSB1C3 (Ligation product)2DH5α1012SOC
9/17 Control (SmaX3, Ligation product)2DH5α1012SOC

Make Control (for 9/18 UV-Spectrum)

Murata

Add MTOB (1.6mg) to ethanenitrile (313.4µL)

Electrophoresis

Yasuda and Honda

(1)

LaneSample
1-29/15 AT-His-T7-RBS His (1)(2)
3-79/15 AT-His-T7-RBS His (1)-(5)
89/16 dddD-T7-RBS(After mutation)(1)
91kb ladder

(2)

LaneSample
11kb ladder
29/16 dddD-T7-RBS(After mutation)(2)
3-89/16 SAMmt-pSB1C3 (1)-(6)

(3)

LaneSample
11kb ladder
29/16 SAMmt-pSB1C3 (7)

Liquid Culture

Yasuda
Sample
9/16 AT His-T7-RBS His (2)-(4)
9/16 SAMmt-pSB1C3 (2)(4)

DNA Purification

Murata
SampleConcentration/(µg/mL)260/280260/230
9/17 REDOX His (X,P)1.61.74-
9/17 REDOX (X,P)(PCR product)1.32.43-

9/18

Miniprep

Murata
SampleConcentration/(µg/mL)260/280260/230
9/15 T7-AT-His (1)2001.541.60
9/15 T7-AT-His (2)1801.642.02
9/16 T7-AT-His (2)1401.842.09
9/16 T7-AT-His (3)1401.711.82
9/16 T7-AT-His (4)1451.832.16
9/16 SAMmt-pSB1C3 (2)2251.641.71
9/16 SAMmt-pSB1C3 (4)1901.752.12

Colony PCR

Honda

Master Mix

Quick Taq/(µL) Fw M13/(µL) Rv M13/(µL) MilliQ/(µL) Total/(µL)
45 1.8 1.8 41.4 90
Predenature Denature Annealing Extension Cooling
94°C 94°C 55°C 68°C 12°C
2min 30sec 30sec 90sec Infinity

Denature-Annealing-Extension 30cycle

Number Sample
1 9/16 REDOX-(1)-1
2 9/16 DiDECARBO-(1)-1
3 9/16 DiDECARBO-(1)-2
4 9/16 DiDECARBO-(1)-3
5 9/16 DiDECARBO-(1)-4
6 9/17 AT-T7-RBS-1
7 9/17 REDOX-His-YCplac111-1
8 9/17 REDOX-His-YCplac111-2
9 9/17 REDOX-His-YCplac111-3
LaneSample
11kb ladder
29/16 REDOX-YCplac111 (1)
3-69/16 DiDECARB-YCplac111 (1)-(4)
LaneSample
1-
21kb ladder
39/17 AT-T7-RBS (1)
4-6REDOX-His=YCplac111 (1)-(3)

Liquid Culture

Honda
SampleMedium
9/17 REDOX-His-YCplac111-(1) CP
9/17 REDOX-His-YCplac111-(2) CP
9/14 AT-pSB1C3-(1) CP

Restriction Enzyme Digestion

Honda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
9/15 AT-pSB1C3-(3) 15 2 - - 2 H 6 6 51.5 82.5
9/15 AT-pSB1C3-(2) 19 - 2 2 - MC 6 6 45 80
9/9 T7-RBS-pSB1C3 14 - - 2 2 B 6 6 50 80
9/18 T7-RBS-AT-His-pSB1C3 33 - - 3 3 B 9 9 63 120

UV-spequtrum measurement

Yasuda and Murata Ethanenitrile solution of saturated MTOB (x10)

Mutation PCR

Honda

SAMmt (SpeI)

Prime STAR MAX Premix(2x) Fw SAMmt SpeI mutation primer Rv SAMmt SpeI mutation primer 9/18 SAMmt-pSB1C3-4 MilliQ Total
25µL 1µL 1µL pick upµL 23µL 50µL
Predenature Denature Annealing Extension Cooling
98°C 98°C 55°C 72°C 12°C
5min 10sec 15sec 30sec Infinity

Denature-Annealing-Extension 30cycle

Quality check of preligation

Murata
SampleConcentration(ng/µL)A260A280260/280260/230
DiDECARB538.3610.7675.7341.882.07
DECARB223.374.4672.3611.891.99
SAMmt-pSB1C3185.243.7051.8961.951.87
REDOX454.079.0814.8001.892.17
AT-pSB1C3299.735.9952.9932.001.99
T7-RBS306.316.1262.9012.111.97
T7-RBS-His307.596.1523.1291.971.50
Pcon-pSB1C3245.884.9182.4232.031.65

PCR

Murata
DiDECARB 10x Buffer 2mM dNTPs 25mM MgSO4 #1 #2 KOD(1U/µL) MilliQ Total
1µL 2.5µL 2.5µL 1.5µL 1.25µL 1.25µL 0.5µL 14.5µL 25

25µL x4 (100µL)


(1)
#1: DiDECARB OE-A (10 µM)
#2: DiDECARB Mutation Rv (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 48sec Infinity

Denature-Annealing-Extension 30cycle


(2)
#1: DiDECARB OE-D (10 µM)
#2: DiDECARB Mutation Fw (10 µM)

(3)
#1: RV-DiDECARB His (10 µM)
#2: DiDECARB Mutation Fw (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 78sec Infinity

Denature-Annealing-Extension 30cycle


(4)
#1: DiDECARB OE-A (10 µM)
#2: DiDECARB MutationP Rv (10 µM)

(5)
#1: DiDECARB MutationP-Fw (10 µM)
#2: DiDECARB MutationE-Rv (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 48sec Infinity

Denature-Annealing-Extension 30cycle


(6)
#1: DiDECARB OE-D (10 µM)
#2: DiDECARB Mutation-E-Fw (10 µM)

(7)
#1: RV-DiDECARB His (10 µM)
#2: DiDECARB Mutation E-Fw (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 78sec Infinity

Denature-Annealing-Extension 30cycle


(8)
#1: SAMmt OE-A (10 µM)
#2: SAMmt Mutation-Rv (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 48sec Infinity

Denature-Annealing-Extension 30cycle


(9)
#1: SAMmt OE-D (10 µM)
#2: SAMmt Mutation-Fw (10 µM)

(10)
#1: RV-SAMmt His (10 µM)
#2: SAMmt Mutation-Fw (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 78sec Infinity

Denature-Annealing-Extension 30cycle


(11)
#1: Fw-Pre-REDPX (10 µM)
#2: Suf-REDOX-RV (10 µM)

(12)
#1: Fw-Pre-REDPX (10 µM)
#2: RV-REDOX-His (10 µM)

Predenature Denature Annealing Extension Cooling
94°C 98°C 50-65°C 68°C 15°C
2min 10sec 30sec 48sec Infinity

Denature-Annealing-Extension 30cycle

Restriction Enzyme Digestion

Murata
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)XhoIDraIBuffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
AT-pSB1C3 (299ng/µL)4-1-111M553250
AT-pSB1C3 (299ng/µL)6.8-1-1--M3315.2030
pSB1C3-T7-RBS (310ng/µL)7--11--H51*3550
Pcon-pSB1C3 (245ng/µL)41--1--H51*3850

The cell marked * is not BSA but BAP.

9/19

Sequence PCR

Honda
Number/(µL) Sample Template/(µL) BigDye/(µL) 5x Buffer/(µL) Primer/(µL) MilliQ/(µL) Conc/(µL)
1 T7-RBS-AT-His(9/16-(2)) 2.8 0.5 1.75 VF 0.5 4.95 10.5
2 T7-RBS-AT-His(9/16-(3)) 2.8 0.5 1.75 VF 0.5 4.95 10.5
3 SAM-pSB1C3(4) 2.8 0.5 1.75 VF 0.5 4.95 10.5
4 T7-RBS-AT-His(9/16-(2)) 2.8 0.5 1.75 VR 0.5 4.95 10.5
5 T7-RBS-AT-His(9/16-(3)) 2.8 0.5 1.75 VR 0.5 4.95 10.5
6 SAM-pSB1C3(4) 2.8 0.5 1.75 VR 0.5 4.95 10.5
7 T7-RBS-AT-His(9/16-(2)) 2.8 0.5 1.75 seg-1 0.5 4.95 10.5
8 T7-RBS-AT-His(9/16-(3)) 2.8 0.5 1.75 seg-1 0.5 4.95 10.5
9 SAM-pSB1C3(4) 2.8 0.5 1.75 OE-B 0.5 4.95 10.5
10 T7-RBS-AT-His(9/16-(2)) 2.8 0.5 1.75 seg-2 0.5 4.95 10.5
11 9/23M PQLB 2.8 0.5 1.75 seg-2 0.5 4.95 10.5
Predenature Denature Annealing Extension Cooling Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min infinity

Denature-Annealing-Extension 30cycle

Gel Extraction

Honda
Lane Sample
1,2,4,5,8 1kbp ladder
3 T7-RBS-AT-His-pSB1C3 S,P
6 T7-RBS-pSB1C3 S,P

Miniprep

Honda
Sample Concentration/(µg/mL) 260/280 260/230
9/18 T7-RBS-AT-His(2) 305 1.66 1.49
9/18 T7-RBS-His-pSB1C3(1) 280 1.68 1.67
9/18 REDOX(His)-YC Plac3(1) 985 1.74 2.39
9/18 REDOX(His)-YC Plac3(2) 1025 1.72 2.28

Gel Extraction

Yasuda
Lane Sample
1,2 9/18 AT-pSB1C3 X,S
3 100bp ladder
5,6 9/18 AT-pSB1C3 E,P

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
9/19 T7-RBS-His Miniprep product 20 - - 3 3 B 12 0 82 120
9/19 REDOX-3 M(1) 10.1 5 - - 5 H 20 0 159.9 200
9/19 REDOX-3 M(1) control 0.1 - - - - H 1 - 8.9 10

Electrophoresis

Honda
Lane Sample
1 DiDECARB-OE-A 52.1°C
2 DiDECARB-OE-A 55.4°C
3 DiDECARB-OE-A 59.4°C
4 DiDECARB-OE-A 64.9°C
5 DiDECARB-OE-A 52.1°C
6 DiDECARB-OE-A 55.4°C
7 DiDECARB-OE-A 59.4°C
8 DiDECARB-OE-A 64.9°C
9 DiDECARB-Mutation PCR product Pst1 Fwd 52.1°C
10 DiDECARB-Mutation PCR product Pst1 Fwd 55.4°C
11 DiDECARB-Mutation PCR product Pst1 Fwd 59.4°C
12 DiDECARB-Mutation PCR product Pst1 Fwd 64.9°C
13 REDOX-Fwd 52.1°C
14 REDOX-Fwd 55.4°C
15 REDOX-Fwd 59.4°C
16 REDOX-Fwd 64.9°C
17 REDOX-Fwd-His 52.1°C
18 REDOX-Fwd-His 55.4°C
19 REDOX-Fwd-His 59.4°C
20 REDOX-Fwd-His 64.9°C
21 DiDECARB-Mutation PCR product Fwd-His 52.1°C
22 DiDECARB-Mutation PCR product Fwd-His 55.4°C
23 DiDECARB-Mutation PCR product Fwd-His 59.4°C
24 DiDECARB-Mutation PCR product Fwd-His 64.9°C
25 DiDECARB-Mutation PCR product Fwd 52.1°C
26 DiDECARB-Mutation PCR product Fwd 55.4°C
27 DiDECARB-Mutation PCR product Fwd 59.4°C
28 DiDECARB-Mutation PCR product Fwd 64.9°C
29 SAM-Mutation PCR product Fwd 52.1°C
30 SAM-Mutation PCR product Fwd 55.4°C
31 SAM-Mutation PCR product Fwd 59.4°C
32 SAM-Mutation PCR product Fwd 64.9°C
33 1kbp ladder
34 SAM-Mutation PCR product Fwd 52.1°C
35 SAM-Mutation PCR product Fwd 55.4°C
36 SAM-Mutation PCR product Fwd 59.4°C
37 SAM-Mutation PCR product Fwd 64.9°C
38 SAM-Mutation PCR product Fwd-His 52.1°C
39 SAM-Mutation PCR product Fwd-His 55.4°C
40 SAM-Mutation PCR product Fwd-His 59.4°C
41 SAM-Mutation PCR product Fwd-His 64.9°C
42 1kbp ladder
43 DiDECARB-Mutation PCR product E-His 52.1°C
44 DiDECARB-Mutation PCR product E-His 55.4°C
45 DiDECARB-Mutation PCR product E-His 59.4°C
46 DiDECARB-Mutation PCR product E-His 64.9°C
47 DiDECARB-Mutation PCR product E-Fwd 52.1°C
48 DiDECARB-Mutation PCR product E-Fwd 55.4°C
49 DiDECARB-Mutation PCR product E-Fwd 59.4°C
50 DiDECARB-Mutation PCR product E-Fwd 64.9°C

PCR

Murata

Template;DiDECARB-OE-A,DiDECARB Mutation product Fw

10x Buffer/(µL) 2.0mM dNTP/(µL) 25.mM MgSO4/(µL) 10.0µM DiDECARB-OE-A/(µL) 10.0µM DiDECARB-OE-D/(µL) 1.0U/µL KOD/(µL) H2O Total/(µL)
10 10 6 5 5 2 up tp 100 100

Template;DiDECARB-OE-A,DiDECARB Mutation product Fw-His

10x Buffer/(µL) 2.0mM dNTP/(µL) 25.mM MgSO4/(µL) 10.0µM DiDECARB-OE-A/(µL) 10.0µM Rv DiDECARB-His/(µL) 1.0U/µL KOD/(µL) H2O Total/(µL)
10 10 6 5 5 2 up tp 100 100

Template;DiDECARB-OE-A,DiDECARB Mutation produc Pst1t Fw,DiDECARB Mutation produc Eco1

10x Buffer/(µL) 2.0mM dNTP/(µL) 25.mM MgSO4/(µL) 10.0µM DiDECARB-OE-A/(µL) 10.0µM DiDECARB-OE-D/(µL) 1.0U/µL KOD/(µL) H2O Total/(µL)
10 10 6 5 5 2 up tp 100 100

Template;DiDECARB-OE-A,DiDECARB Mutation product Pst1 Fw,DiDECARB Mutation product Eco1-His

10x Buffer/(µL) 2.0mM dNTP/(µL) 25.mM MgSO4/(µL) 10.0µM DiDECARB-OE-A/(µL) 10.0µM Rv DiDECARB-His/(µL) 1.0U/µL KOD/(µL) H2O Total/(µL)
10 10 6 5 5 2 up tp 100 100

Template;SAM-OE-A,SAM Mutation product Fw

10x Buffer/(µL) 2.0mM dNTP/(µL) 25.mM MgSO4/(µL) 10.0µM SAM-OE-A/(µL) 10.0µM SAM-OE-D/(µL) 1.0U/µL KOD/(µL) H2O Total/(µL)
10 10 6 5 5 2 up tp 100 100

Template;SAM-OE-A,SAM Mutation product Fw-His

10x Buffer/(µL) 2.0mM dNTP/(µL) 25.mM MgSO4/(µL) 10.0µM SAM-OE-A/(µL) 10.0µM SAM His/(µL) 1.0U/µL KOD/(µL) H2O Total/(µL)
10 10 6 5 5 2 up tp 100 100
Sample Concentration/(µg/mL) 260/280 260/230
pSB1C3-AT/Pst1,Xba1,Xhol1,Dra1 24.75 1.90 1.92
T7RBS/Spe1,Pst1 8.48 1.91 1.01
T7RBS-His/Spe1,Pst1 35.98 1.88 1.78

Gel Extraction/Electrophoresis

Murata
Lane Sample
2~5 DiDECARB His
6~8,10 DiDECARB
11~14 SAM
16~20 SAM His
21~23,25 DE His
26~30 DE
1,9,15,24 Blank

DNA Purification

Murata

DiDECARB His

DiDECARB

SAM

SAM His

Restriction Enzyme Digestion

Murata
Sample/(ng) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
DiDECARB His X,P 2000 - 1 - 1 M 3 3 up tp 30 30
DiDECARB X,P 2000 - 1 - 1 M 3 3 up to 30 30
DiDECARB E,P 2000 1 - - 1 H 3 - up to 30 30
SAM X,P 2000 - 1 - 1 M 3 3 - 30
SAM E,P 2000 1 - - 1 H 3 - up to 30 30
SAM His X,P 2000 - 1 - 1 M 3 3 up to 30 30

9/20

Electrophoresis

Murata
Lane Sample
1 SAM His X,P
2 Di X,P
3 Di His X,P
4 SAM E,P
5 SAM X,P
6 Di E,P
7 AT X,P 2
Lane Sample
1 AT X,P 3
2 T7-RBS S,P
3 dddD Mutation PCR product (2)
4 dddD Mutation PCR product (1)
5 T7-RBS pSB1C3
6 REDOX PCR product
7 REDOX His PCR product

Transformation

Murata
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
7-2 DiDECARB T7-RBS 2 DH5α 10 12 SOC
7-5 SAMmt T7-RBS 2 DH5α 10 12 SOC
7-11 T7-RBS dddD(1) Mutation PCR product 2 DH5α 10 12 SOC
7-12 T7-RBS dddD(3) Mutation PCR product 2 DH5α 10 12 SOC
7-13 T7-RBS AT 2 DH5α 10 12 SOC
T7-RBS control 2 DH5α 10 12 SOC
10-1 DiDECARB pSB1C3 2 DH5α 10 12 SOC
10-4 SAMmt pSB1C3 2 DH5α 10 12 SOC
10-8 REDOX pSB1C3 2 DH5α 10 12 SOC
pSB1C3 control 2 DH5α 10 12 SOC
14-3 T7-RBS SAM-His 2 DH5α 10 12 SOC
T7-RBS DiDECARB His 2 DH5α 10 12 SOC
REDOX T7 RBS His(14)-9 2 DH5α 10 12 SOC
T7-RBS-His control 2 DH5α 10 12 SOC

9/22

Colony PCR

Yasuda
Quick Tag/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension Cooling
94°C 94°C 55°C 68°C 12°C
2min 30sec 30sec 150sec infinity

Denature-Annealing-Extension 30cycle

Number Sample
1~8 REDOX-T7-RBS-His
9~16 T7-RBS-SAM-His
17~24 T7-RBS control
25~32 T7-RBS-dddD
33~40 DiDECARB-pSB1C3
41~48 pSB1C3 control
49~56 T7-RBS-DiDECARB
57~64 T7-RBS-His
65~72 REDOX-pSB1C3
73~80 DiDECARB-T7-RBS
81~88 SAM-pSB1C3
89~96 T7-RBS-sssS
97~104 T7-RBS-SAM
105 T7-RBS-AT
106~123 pBC345-AT

Colony PCR

Honda

Master Mix

Quick Tag/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
225 9 9 217 450
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 150sec

Denature-Annealing-Extension 30cycle

Number Sample
1~7 9/20 T7-RBS-REDOX-His
8~14 9/20 SAMmt-pSB1C3
15~21 9/20 T7-RBS-SAMmt-His
22~28 9/20 DiDECARB-pSB1C3
29~35 9/20 T7-RBS-DiDECARB
36~42 9/20 T7-RBS-DiDECARB-His
43 9/20 T7-RBS-dddD
44 9/20 DiDECARB-pSB1C3

Miniprep

Tatsui
Sample Concentration/(µg/mL) 260/280 260/230
T7-RBS-His 55 1.58 0.80

Concentration Measurement

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
T7-RBS-His S,P 1.0 1.54 1.94
T7-RBS-His S,P 1.3 1.59 2.01
T7-RBS E,S 1.1 1.40 2.01
HT-pSB1C3 X,P 4.4 1.52 1.08
AT-pSB1C3 X,P 4.0 1.65 1.06

Cut Check

Yasuda
Lane Sample
1 Blank
2 100bp GeneRuler
3 T7 RBS-His S,P
4 T7-RBS S,P
5 T7-RBS E,P
6 AT-pSB1C3 X,P
7 AT-pSB1C3 X,P

Electrophoresis

Honda

(1)

Lane Sample
1 100b ladder
2~8 T7-RBS-REDOX-His

(2)

Lane Sample
1 100b ladder
2~8 SAMmt-pSB1C3

(3)

Lane Sample
1 100b ladder
2~8 T7-RBS-SAMmt-His

(4)

Lane Sample
1 100b ladder
2~8 DiDECARB-pSB1C3

(5)

Lane Sample
1 100b ladder
2~8 T7-RBS-DiDECARB

(6)

Lane Sample
1 100b ladder
2~8 T7-RBS-DiDECARB-His

(7)

Lane Sample
1 Blank
2 Blank
3 100b ladder
4 T7-RBS-dddD-1
5 DiDECARB-pSB1C3-1

Liquid Culture

Yasuda
Sample Medium
T7-RBS-dddD CP
Sample Medium
REDOX-pSB1C3 CP
Sample Medium
T7-RBS-SAMmt CP

Colony PCR

Honda

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
240 9.6 9.6 220.8 480
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 90sec

Denature-Annealing-Extension 30cycle

Number Sample
1~16 T7-RBS-DiDECARB-His
19~32 T7-RBS-SMAmt-His
33~48 T7-RBS-REDOX-His

9/23

Miniprep

Miniprep
Sample Concentration/(µg/mL) 260/280 260/230
pB-22 250 1.61 1.69
pB-23 125 2.07 2.90
pQ-3 185 1.68 1.87
pQ-5 210 1.72 1.86
pLQB 90 2.40 3.09

Electrophoresis

Honda

1

Number Sample
1 100bp ladder
2~8 T7-RBS-REDOX-His 2~7

2

Number Sample
1 100bp ladder
2~8 T7-RBS-REDOX-His 8~14

3

Number Sample
1 100bp ladder
2,3 T7-RBS-REDOX-His 15,16
4~8 T7-RBS-SAMmt-His 1~5

4

Number Sample
1 100bp ladder
2~8 T7-RBS-SAMmt-His 6~12

5

Number Sample
1 100bp ladder
2~5 T7-RBS-SAMmt-His 13~16
6~8 T7-RBS-DiDECARB-His 1~3

6

Number Sample
1 100bp ladder
2~8 T7-RBS-DiDECARB-His 4~10

7

Number Sample
1 100bp ladder
2~7 T7-RBS-DiDECARB-His 11~16

9/24

Dephosphorylation

Yasuda

T7-RBS-His S,P

Miniprep

Murata
Sample Concentration/(µg/mL) 260/280 260/230
T7-RBS-SAM 6 10.3 1.67 1.15
9/22 DiDECARB pSB1C3-4 11.3 1.54 1.11
9/22 DiDECARB pSB1C3-2 10.5 1.67 0.83
9/20 T7-RBS-dddD Mutation PCR product(3)-(4) 14.2 1.71 1.38
REDOX-pSB1C3(2) 9.8 1.71 0.83
9/20 T7-RBS-dddD Mutation PCR product(3)-(5) 12.7 1.66 1.42
9/22 DiDECARB pSB1C3-5 10.5 1.69 1.15

Cycle PCR

Yasuda

Master Mix

Number/(µL) Sample Template/(µL) BigDye/(µL) 5x Buffer/(µL) Primer/(µL) MilliQ/(µL) Conc/(µL)
1 9/23M PQLB 4.4 0.5 1.75 VF2 0.5 2.8 90
2 9/23M PQLB 4.4 0.5 1.75 pQLB seq-1 0.5 2.8 90
3 9/23M PQLB 4.4 0.5 1.75 pQLB seq-2 0.5 2.8 90
4 9/23M PQLB 4.4 0.5 1.75 pQLB seq-3 0.5 2.8 90
5 9/23M PQLB 4.4 0.5 pQLB seq-4 VF2 0.5 2.8 90
6 9/23M PQLB 4.4 0.5 1.75 VR 0.5 2.8 90
7 9/23M PQ-3 2.2 0.5 1.75 VF2 0.5 5.0 185
8 9/23M PQ-3 2.2 0.5 1.75 VR 0.5 5.0 185
9 9/23M PQ-5 1.9 0.5 1.75 VF2 0.5 5.3 210
10 9/23M PQ-5 1.9 0.5 1.75 VR 0.5 5.3 210
11 9/22M pB-23 3.2 0.5 1.75 VF2 0.5 4.0 125
12 9/22M pB-23 3.2 0.5 1.75 VR 0.5 4.0 125
13 9/23M pB-22 1.6 0.5 1.75 VF2 0.5 5.7 250
14 9/23M pB-22 1.6 0.5 1.75 VR 0.5 5.7 250
15 9/15 MP AT-pSB1C3 1 1.3 0.5 1.75 VF2 0.5 6.0 299
16 9/15 MP AT-pSB1C3 1 1.3 0.5 1.75 AT Seq1 0.5 6.0 299
17 9/15 MP AT-pSB1C3 1 1.3 0.5 1.75 AT Seq2 0.5 6.0 299
18 9/15 MP AT-pSB1C3 1 1.3 0.5 1.75 VR 0.5 6.0 299
19 9/22 M REDOX pSB1C3 0.8 0.5 1.75 VF2 0.5 6.4 490
20 9/22 M REDOX pSB1C3 0.8 0.5 1.75 REDOX Seq1 0.5 6.4 490
21 9/22 M REDOX pSB1C3 0.8 0.5 1.75 VR 0.5 6.4 490
22 9/18 MP SAM-pSB1C3 2.1 0.5 1.75 VF2 0.5 5.0 185
23 9/18 MP SAM-pSB1C3 2.1 0.5 1.75 SAM Seq1 0.5 5.0 185
24 9/18 MP SAM-pSB1C3 2.1 0.5 1.75 VR 0.5 5.0 185
25 9/22 DiDECARB pSB1C3-4 0.7 0.5 1.75 VF 0.5 6.5 565
26 9/22 DiDECARB pSB1C3-4 0.7 0.5 1.75 DiDECARB Seq1 0.5 6.5 565
27 9/22 DiDECARB pSB1C3-4 0.7 0.5 1.75 DiDECARB Seq2 0.5 6.5 565
28 9/22 DiDECARB pSB1C3-4 0.7 0.5 1.75 VR 0.5 6.5 565

DNA Purification

Honda
Sample Concentration/(µg/mL) 260/280 260/230
9/24 PCR product AT 265 1.67 2.10

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
9/24 REDOX(His) column 7.5 - 1 - 1 M 3 3 14.5 30

9/25

Colony PCR

Honda
Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension Cooling
94°C 94°C 55°C 68°C 12°C
2min 30sec 30sec 1min infinity

Denature-Annealing-Extension 30cycle

Number Sample
1 T7-RBS-REDOX-His(1)
2 T7-RBS-REDOX-His(2)
3 T7-RBS-REDOX-His(3)

Electrophoresis

Honda
Lane Sample
1,2 Blank
3 100b ladder
4~6 T7-RBS-REDOX-His(1)~(3)

Colony PCR

Yasuda

Master Mix

Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
130 5.2 5.2 119.6 260.0
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 90sec

Denature-Annealing-Extension 30cycle

Number Sample
1~8 T7-RBS-REDOX-His (1)~(8)
9~16 T7-RBS-SAMmt-His (1)~(8)
17~24 T7-RBS-DiDECARB-His (1)~(8)

Electrophoresis

Yasuda

1

Lane Sample
1 100bp GeneRuler
2~8 T7-RBS-REDOX-His(1)~(7)

2

Lane Sample
1 100bp GeneRuler
2 T7-RBS-REDOX-His(8)
3~7 T7-RBS-SAMmt-His(1)~(5)
8 Blank

3

Lane Sample
1 100bp GeneRuler
2~4 T7-RBS-SAMmt-His(6)~(8)
5~8 T7-RBS-DiDECARB-His(1)~(4)

4

Lane Sample
1 100bp GeneRuler
2,3,5,6 T7-RBS-DiDECARB-His(5)~(8)
4 Blank

Transformation

Yasuda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
T7-RBS-AT-His 9/19 M(3) 1 Zip BL21(DE3) 10 11 CP
T7-RBS-dddD(n) 9/22 M (3)-5 1 Zip BL21(DE3) 10 11 CP

9/26

Colony PCR

Yasuda
Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension Cooling
94°C 94°C 55°C 68°C 12°C
5min 30sec 30sec 2min infinity

Denature-Annealing-Extension 30cycle

Number Sample
1~8 T7-RBS-REDOX-His
9~16 T7-RBS-SAMmt-His
17~24 T7-RBS-DiDECARB-His

Electrophoresis

Yasuda
Lane Sample
1~8 T7-RBS-REDOX-His(1)~(8)
9~12,14~17 T7-RBS-SAMmt-His(1)~(8)
18~25 T7-RBS-DiDECARB-His(1)~(8)
13 100bp ladder

9/27

Colony PCR

Yasuda
Quick Taq/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 160sec

Denature-Annealing-Extension 30cycle

Number Sample
1~7 T7-RBS-REDOX-His
8~14 T7-RBS-SAMmt-His
15~20 T7-RBS-DiDECARB-His
21,22 T7-RBS-AT-His 9/25 Transformation(3),(4)
23,24 T7-RBS-dddD 9/25 Transformation(3),(4)

Cloning PCR

Yasuda

Master Mix

KOD Plus/(µL) DNA CCAP 1023/11/(µL) 2mMdNTPs/(µL) 10xBuffer forKDDplusV2/(µL) 25mM MgSO4/(µL) Fw AT cloning primer 61/(µL) Rv AT cloning primer 61/(µL) MilliQ/(µL) Total/(µL)
1 2 5 5 3 1.5 1.5 31 50
Predenature Denature Annealing Extension Cooling
94°C 98°C 56°C 68°C 12°C
2min 10sec 30sec 90sec infinity

Denature-Annealing-Extension 30cycle

9/28

OE-PCR 1st

Yasuda
KOD Plus/(µL) 9/27 AT PCR product/(µL) 10xBuffer for KODPlus/(µL) 2mMdNTPS/(µL) 25mM MgSO4/(µL) Primer #1/(µL) MilliQ/(µL) Total/(µL)
1 pick up 5 5 3 1.5(each) up to 50 50

1. #1 AT OE-A/AT OE-B

2. #1 AT OE-C/AT2 OE-B

3. #1 AT2 OE-C/AT OE-D

Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 55°C 72°C 72°C 12°C
2min 10sec 10sec 1min 5min infinity

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
8/27 Miniprep product 1A2 7.6 - - 3 3 B 12 1.2 93.2 120
control 0.5 - - - - B 1 0.1 8.4 10

Gel Extraction

Yasuda
Lane Sample
1 9/28 OE-(3)
2 9/28 OE-(3)
3 Blank
4 9/28 OE-(1)
5 100bp ladder
Lane Sample
1 9/28 OE-(3)
4 9/28 OE-(2)
6 100bp ladder
2,3,5 Blank

OE-PCR 2nd

Yasuda

Master Mix

KOD Plus/(µL) 10xBuffer for KODPlus ver2/(µL) 2mMdNTPS/(µL) 25mM MgSO4/(µL) Primer #1/(µL) Template #2/(µL) MilliQ/(µL) Total/(µL)
1 5 5 3 2(each) 0.5(each) up to 50 50

1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product

2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column

Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 55°C 72°C 72°C 12°C
2min 10sec 10sec 1min 5min infinity

Denature-Annealing-Extension 25cycle

9/29

Mutation PCR

Yasuda
PrimeStar Max Premix/(µL) SAMmt Mutation Fw/(µL) SAMmt Mutation Rv/(µL) 9/18 MP SAM-pSB1C3/(µL) MilliQ/(µL) Total/(µL)
25 1 1 0.5 22.5 50
Denature Annealing Extension
98°C 55°C 72°C
10sec 15sec 55sec

Denature-Annealing-Extension 30cycle

Gel Extraction

Yasuda
Lane Sample
5 100bp ladder
3,7 1A2 9/28 S,P
9 9/28 Digestion control
1,2,4,5,6,8 Blank

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
8/27 M 1A2 7.6 - 3 - 3 D 12 1.2 93.2 10
control 0.5 - - - - D 1 0.1 8.4 10

Gel Extraction

Yasuda
Lane Sample
1 100bp ladder
4 1A2 9/29 X,P
6 9/29 Digestion control
2,3,5 Blank

Concentration Measurement

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
1. 9/28 OE(1) 9/29 gel extraction product 17.9 1.92 0.94
2. 9/28 OE(2) 9/29 gel extraction product 122.2 1.86 1.64
3. 9/28 OE(3) 9/29 gel extraction product 78.8 1.89 1.67
1A2 9/29 X,P 26.1 1.37 0.76

Transformation

Murata
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
9/29 Ligation DiDECARB(His)-1A2 1 Zip BL21(DE3) 10 11 CP
9/29 Ligation REDOX(His)-1A2 1 Zip BL21(DE3) 10 11 CP
9/29 SAMmt mutation 1 Zip BL21(DE3) 10 11 CP

9/30

Mutation PCR

Yasuda
PrimeStar Max PreMix/(µL) Template #1/(µL) Primers #2/(µL) MilliQ/(µL) Total/(µL)
25 0.5 1(each) up to 50 50

1. #1 9/15 Miniprep AT-pSB1C3(1) #2 Fw:AT Mutation Insert/Rv:AT Mutation Insert

2. #1 9/19 Miniprep T7-RBS-AT(His) #2 Fw Add His to T7RBS/Rv Add His toT7 RBS

3. #1 9/13 Miniprep T7-RBS-pSB1C3 #2 Fw Add His to T7RBS/Rv Add His to T7RBS

Denature Annealing Extension Cooling
98°C 55°C 72°C 12°C
10sec 15sec 20sec infinity

Denature-Annealing-Extension 35cycle

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
SAMmt-pSB1C3 9/30 column 136.3 1.87 1.68

OE-PCR 2nd

Yasuda

Master Mix

KOD Plus/(µL) 10xBuffer for KODPlus ver2/(µL) 2mMdNTPS/(µL) 25mM MgSO4/(µL) Primer #1/(µL) Template #2/(µL) MilliQ/(µL) Total/(µL)
1 5 5 3 2(each) 0.5(each) up to 50 50

1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product

2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column

Predenature Denature Annealing Extension Final Extension Cooling
98°C 98°C 55°C 72°C 72°C 12°C
2min 10sec 10sec 1min 5min infinity

Denature-Annealing-Extension 30cycle

Transformation

Yasuda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
T7-RBS-AT-His 1 self-making DH5α 10 11 CP
SAMmt-pSB1C3 9/29 Mutation PCR product 1 self-making DH5α 10 11 CP
AT-pSB1C3 9/30 insertion Mutation PCR product 1 self-making DH5α 10 11 CP
T7-RBS-His 9/30 Mutation PCR product 1 self-making DH5α 10 11 CP

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
9/30 OE-PCR 2nd product(Pre-AT-Suf) 217 1.85 1.22

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
Pre-At-Suf 9/30 Column 9.2 1 - - 1 H 4 0.4 14.4 40

10/1

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
AT 9/30 E,P 38 1.84 0.46
pSB1C3 E,P 5 1.78 0.27

Transformation

Yasuda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
9/30 AT-pSB1C3 1 BL21(DE3) 10 11 CP
9/30 T7-RBS-His 1 self-making DH5α 2/23 10 11 CP
9/30 T7-RBS-AT-His 1 self-making DH5α 2/23 10 11 CP
9/29 SAMmt-pSB1C3 1 self-making DH5α 2/23 10 11 CP
9/22 T7-RBS-dddD(3)-5 1 self-making DH5α 2/23 10 11 CP

10/2

Kination

Yasuda

Add His to T7RBS Fw

ATOE-D

PCR

Yasuda
PrimeSTAR Premix/(µL) Template #1/(ng) Primer #2/(µL) MilliQ/(µL) Total/(µL)
50 400 2(each) up to 100 100

#1 AT-pSB1C3(without (1))

#2 Fw:Add His to T7RBS/Rv:AT OE-D

DNA Purification

Murata
Sample
PCR product (1),(2) His-pSB1C3-AT
PCR product(3) AT-pSB1C3 (AT Mota I)
PCR product(4) AT-pSB1C3 (AT Mota D)
PCR product(5) T7-RBS-His-pSB1C3

Restriction Enzyme Digestion

Murata
Dpn1/(µL) 10/2 PCR AT-pSB1C3 miniprep product(3)/ (µL) 10x Buffer(H)/(µL) MilliQ/(µL) Total/(µL)
1 8 5 36 50

Electrophoresis

Yasuda
Lane Sample
4 His pSB1C3-AT 10/2 DNA Purification
5 AT-pSB1C3 Mutation Deletion 10/2 DNA Purification
6 1kbp GeneRuler
7 AT-pSB1C3 Mutation Insert 10/2 DNA Purification
8 T7-RBS AT His 10/2 DNA Purification
1~3,9~12 Blank

10/3

Miniprep

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
9/14 AT-pSB1C3(1) 119 2.00 2.19
10/1 SAMmt(Mutated)-pSB1C3(1) 275 1.86 1.75
10/1 SAMmt(Mutated)-pSB1C3(2) 227 1.93 1.90
10/1 T7-RBS-His(1) 147 2.00 1.64
10/1 T7-RBS-His(2) 107 1.94 1.74

Mutation PCR

Murata

Master Mix

PrimeSTARPremix/(µL) Template 10/3 Miniprep AT-pSB1C3(1)/(µL) Primer AT Mutation Insert Rv/(µL) AT Mutation Insert Fw/(µL) MilliQ/(µL) Total/(µL)
12.5 119 0.5 0.5 up to 25 25
Denature Annealing Extension
94°C 55°C 68°C
10sec 15sec 20sec

Denature-Annealing-Extension 35cycle

Gel Extraction

Yasuda
Lane Sample
1 His-OE-(1)
2 His-OE-(2)

Electrophoresis

Yasuda
Lane Sample
1 AT-pSB1C3 Mutation insert 50.2°C
2 AT-pSB1C3 Mutation insert 52.8°C
3 AT-pSB1C3 Mutation insert 54.2°C
4 AT-pSB1C3 Mutation insert 55.7°C
5 AT-pSB1C3 Mutation insert 57.1°C
6 10/2 DNA Purification His pSB1C3-AT
7 AT-pSB1C3 Mutation Deletion 50.2°C
8 AT-pSB1C3 Mutation Deletion 52.8°C
9 AT-pSB1C3 Mutation Deletion 54.2°C
10 AT-pSB1C3 Mutation Deletion 55.7°C
11 AT-pSB1C3 Mutation Deletion 57.1°C
12 100bp ladder

OE-PCR 1st

Murata

(1)

KOD Plus/(µL) 10xBuffer for KOD Plus/(µL) 2mM dNTPs/(µL) 25mM MgSO4/(µL) 10/2 MP J04450(pSB1C3)/(µL) VF2/(µL) Rv mutation primer T7-RBS-His/(µL) MilliQ/(µL) Total/(µL)
1 5 5 3 1 1.5 1.5 32 50

(2)

KOD Plus/(µL) 10xBuffer for KOD Plus/(µL) 2mM dNTPs/(µL) 25mM MgSO4/(µL) 10/2 MP J04450(pSB1C3)/(µL) Fw mutation primer T7-RBS-His/(µL) Present from Santa/(µL) MilliQ/(µL) Total/(µL)
1 5 5 3 1 1.5 1.5 32 50
Denature Annealing Extension Cooling Cooling
94°C 98°C 55°C 68°C 4°C
2min 10sec 30sec 3min infinity

Denature-Annealing-Extension 35cycle

OE-PCR 2nd

Yausda
KOD Plus/(µL) 10xBuffer for KOD Plus/(µL) 2mM dNTPs/(µL) 25mM MgSO4/(µL) His OE-(1) 10/3 gel extraction product/ (µL) His OE-(2) 10/3 gel extraction product/ (µL) VF2/(µL) Present from Santa/(µL) MilliQ/(µL) Total/(µL)
1 5 5 3 1 1 1.5 1.5 31 50
Denature Annealing Extension Cooling Cooling
94°C 98°C 55°C 68°C 4°C
2min 10sec 30sec 3min infinity

Denature-Annealing-Extension 35cycle

Restriction Enzyme Digestion

Yasuda
Sample/(µL) Xba1/(µL) ApnL1/(µL) 10XT/(µL) BSA/(µL) CIAP/(µL) MilliQ/(µL) Total/(µL)
AT-pSB1C3(1) 10/3 Miniprep 10 1 1 4 4 1 19 40
OE-PCR 2nd(T7 RBS His) without CIAP 10 1 1 4 4 - 20 40

Ligation

Yasuda

A

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 10/3 XA 2 T7 RBS His 10/3 XA 2 2xRapidLigBuffer 5 Promega T4 Ligase 1 - 10
pSB1C3 10/3 XA 2 T7 RBS His 10/3 XA - 2xRapidLigBuffer 5 Promega T4 Ligase 1 2 10

B

Vector/(µL) Insert/(µL) Buffer/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 10/3 XA 2 T7 RBS His 10/3 XA 2 2xRapidLigBuffer 4 - 8
pSB1C3 10/3 XA 1 T7 RBS His 10/3 XA - 2xRapidLigBuffer 4 1 6

Transformation

Yasuda
Sample CompetentCells Medium
10/3 Ligation products DH5α CP

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
10/3 OE-PCR 2nd 204.5 1.82 2.33

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
10/3 digestion product(1) 29.6 1.53 1.11
10/3 digestion product(2) 23.2 1.67 0.68

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
RFP OE-1 33.4 1.58 1.08
RFP OE-2 51.7 1.70 1.19

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
10/3 OE-PCR 2nd 416.3 1.81 2.31

Transformation

Yasuda
Sample CompetentCells
10/4 Ligation product DH5α

10/4

Gel Extraction

Yasuda
Sample
10/4 Digestion product

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 10/4 XA 2 RFP His 10/4 XA 2 2x Rapid Ligation Buffer 5 Promega T4 Ligase 1 - 10
pSB1C3 10/4 XA 3 RFP His 10/4 XA 1 2x Rapid Ligation Buffer 5 Promega T4 Ligase 1 - 10
pSB1C3 10/4 XA 1 RFP His 10/4 XA 3 2x Rapid Ligation Buffer 5 Promega T4 Ligase 1 - 10
pSB1C3 10/4 XA 1 RFP His 10/4 XA 1 2x Rapid Ligation Buffer 5 Promega T4 Ligase 1 2 10

10/5

OE-PCR 2nd

Yasuda
KOD Plus/(µL) 10xBuffer for KOD Plus/(µL) 2mM dNTPs/(µL) 25mM MgSO4/(µL) Primers #1/(µL) Templates #2/(µL) MilliQ/(µL) Total/(µL)
2 10 10 6 3(each) 2(each) 62 100

1. #1 VF2/Present from Santa #2 RFP OE(1)/RFP OE(2)

2. #1 VF2/Present from Santa #2 RFP OE(1)/His OE(2)

Denature Annealing Extension Final Extension
94°C 98°C 55°C 68°C
2min 10sec 30sec 100min

Denature-Annealing-Extension 35cycle

Restriction Enzyme Digestion

Yasuda
Sample/(µL) Xba1/(µL) Spe1/(µL) CIAP/(µL) 10XT/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
10/5 RPFHis(1) 10/5 OE2(1) 5 1 1 - 4 4 20 40
10/5 RPFHis(2) 10/5 OE2(2) 5 1 1 - 4 4 20 40
10/5 RPFHis(3) 10/4 OE2 RPF His 5 1 1 - 4 4 20 40
10/5 pSB1C3 XA (1) 9/18 Mutated Bn-pSB1C310-3 5 1 1 1 4 4 19 40
10/5 pSB1C3 XA (2) 9/14 Mutated dddD(mutated)-pSB1C3(2) 5 1 1 1 4 4 19 40

Gel Extraction

Yasuda
Lane Sample
(1)-1,(2)-6 SKY
(1)-2,3 12FP His 10/5 XA(1)
(1)-4,5 pSB1C3 10/5 XA(2)
(1)-6 RFP His 10/5 XA(2)
(2)-1 RFP His 10/5 XA(2)
(2)-2,3 RFP His 10/5 XA(3)
(2)-4,5 pSB1C3 10/5 XA(1)

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
10/5 RFP His1 205.9 1.87 2.39
10/5 RFP His2 157.6 1.84 2.34

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 10/4 XA 1 RFP His 10/4 XA 1 TOYOBO Ligation High 2 - - - 4
- - RFP His 10/4 XA 1 TOYOBO Ligation High 2 - - 1 4

Transformation

Yasuda
Sample CompetentCells
10/5 Ligation product DH5α

Liquid Culture

Yasuda
Sample
10/4 C1
10/4 C2

Gel extraction

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
RFP His XA1 169.8 1.85 1.13
RFP His XA3 62.6 1.79 1.11
pSB1C3 XA 2x 26.0 1.71 1.01

10/6

Miniprep

Honda
Sample Concentration/(µg/mL) 260/280 260/230
10/5 Pcon RPF-His A 133 1.93 2.24
10/5 Pcon RPF-His B 133 1.90 1.83
10/5 Pcon RPF-His C 137 1.80 1.72
10/5 Pcon RPF-His D 142 1.98 2.61

PCR

Honda

10/6 Pcon RPFHisA VF

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.9 0.5 1.75 0.5 4.85 10.5

10/6 Pcon RPFHisA VR

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.9 0.5 1.75 0.5 4.85 10.5

10/6 Pcon RPFHisB VF

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
3.0 0.5 1.75 0.5 4.75 10.5

10/6 Pcon RPFHisB VR

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
3.0 0.5 1.75 0.5 4.75 10.5

10/6 Pcon RPFHisC VF

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.9 0.5 1.75 0.5 4.85 10.5

10/6 Pcon RPFHisC VR

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.9 0.5 1.75 0.5 4.85 10.5

10/6 Pcon RPFHisD VF

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.8 0.5 1.75 0.5 4.95 10.5

10/6 Pcon RPFHisD VR

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.8 0.5 1.75 0.5 4.95 10.5
Predenature Denature Annealing Extension Cooling Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min infinity

Denature-Annealing-Extension 30cycle

10/7

PCR

Honda

AT(1)

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
4.0 0.5 1.75 0.5 3.25 10.5

AT(2)

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.0 0.5 1.75 0.5 5.25 10.5

AT(3)

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.0 0.5 1.75 0.5 5.25 10.5

AT(4)

Template/(µL) Bigdye/(µL) 5xBuffer/(µL) Primer/(µL) MilliQ/(µL) Total/(µL)
2.5 0.5 1.75 0.5 4.75 10.5
Predenature Denature Annealing Extension Cooling Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min infinity

Denature-Annealing-Extension 30cycle

Restriction Enzyme Digestion

Honda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
10/6 T7-RBS-His(2) 12 - - 1 1 B 4 0.4 21.6 40
10/6 T7-RBS-His(2) control 1 - - - - B 1 - 8 10
10/6 T7-RBS-His(3) 20 - - 2 2 B 8 0.8 47.2 80
10/6 T7-RBS-His(3) control 1 - - - - B 1 - 8 10

Gel Extraction

Honda
Lane Sample
1 T7RBSHis(3) 10/7 S,P control
2 T7RBSHis(3) 10/7 S,P Experimental
3 100bp ladder
4 Blank
5 7RBSHis(2) 10/7 S,P Experimental
6 7RBSHis(2) 10/7 S,P control
Lane Sample
3 T7RBSHis(3) 10/7 S,P Experimental
1,2,4,5,6 Blank
Sample Concentration/(µg/mL) 260/280 260/230
T7RBSHis(3) low 38 1.52 1.30
T7RBSHis(2) 46 1.49 1.53
T7RBSHis(3) high 28 2.04 0.38

10/8

Liquid Culture

Honda
Sample Medium
BL21 LB

Liquid Culture

Yasuda
Sample Medium
9/30 AT-pSB1C3 insertion mutated CP
9/30 SAMmt-pSB1C3 mutated CP

10/10

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
AT(Mutation Insert)-pSB1C3(2) 20 - 1 - 1 D 4 0.4 3.6 40

Dephosphorylation

Yasuda
10/8 T7 RBSHis(2)S,P/(µL) CIAP/(µL) 10xBuffer/(µL) MilliQ/(µL) Total/(µL)
12 1 2 5 20
37°C 50°C 15°C
30min 15min 30min

Miniprep

Yasuda
Sample
Liquid Culture product SAM(Mutated)-pSB1C3(1)~(4)

Cloning PCR

Yasuda
2X KAPA HiFI HotStart ReadyMix/(µL) AT OE-A/(µL) AT cloning for His/(µL) 10/7 M AT(Mutation Insert)-pSB1C3(2)/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 up to 25 11 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 60°C 72°C 72°C
5min 20sec 15sec 90sec 5sec

Denature-Annealing-Extension 35cycle

Gel Extraction

Yasuda
Lane Sample
1 AT pSB1C3 (Mutation Insert)(2)10/3
2 Blank
3 100bp ladder

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(1) 10 - - - 1 E 4 0.4 24.6 40
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(2) 10 - - - 1 E 4 0.4 24.6 40
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(3) 10 - - - 1 E 4 0.4 24.6 40
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(4) 10 - - - 1 E 4 0.4 24.6 40

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
T7 RBS-pSB1C3 S,P 2 10/10 gel extraction product(true)XP 2 2xRapidLigaseBuffer 5 Promega T4 Ligase 1 - 10
T7 RBS-pSB1C3 S,P 1 10/10 gel extraction product(true)XP 3 2xRapidLigaseBuffer 5 Promega T4 Ligase 1 - 10
T7 RBS-pSB1C3 S,P 2 10/10 gel extraction product(true)XP 0 2xRapidLigaseBuffer 5 Promega T4 Ligase 1 2 10
T7 RBS-pSB1C3 S,P 2 10/10 gel extraction product(true)XP 2 2xRapidLigaseBuffer 5 Promega T4 Ligase 1 - 10
T7 RBS-pSB1C3 S,P 1 10/10 gel extraction product(true)XP 3 2xRapidLigaseBuffer 5 Promega T4 Ligase 1 - 10
T7 RBS-pSB1C3 S,P 2 10/10 gel extraction product(true)XP 0 2xRapidLigaseBuffer 5 Promega T4 Ligase 1 2 10

Transformation

Yasuda
Sample CompetentCells Medium
10/10 Ligation product BL21(DE3) CP

10/11

Miniprep

Honda
Sample Concentration/(µg/mL) 260/280 260/230
T7RBSHis(2) 217.4 1.92 2.16
T7RBSHis(3) 418.0 1.95 2.30
AT-pSB1C3(2) 258.0 1.96 2.25

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) CIAP/(µL) MilliQ/(µL) Total/(µL)
10/11 miniprep product T7RBSHis(2) 10 - - 2 2 B 8 0.8 47.2 80
10/11 miniprep product T7RBSHis(3) 10 - - 2 2 B 8 0.8 57.2 80
10/11 miniprep product AT-pSB1C3(2) 10 - 1 - 1 B 4 0.4 0 23.6 40
10/10 miniprep product SAM(mutated)-pSB1C3-(1) 10 - - 1 - B 4 0.4 0 24.6 40
10/10 miniprep product SAM(mutated)-pSB1C3-(2) 10 - - 1 - B 4 0.4 0 24.6 40
10/10 miniprep product SAM(mutated)-pSB1C3-(3) 10 - - 1 - B 4 0.4 0 24.6 40
10/10 miniprep product SAM(mutated)-pSB1C3-(4) 10 - - 1 - B 4 0.4 0 24.6 40

DNA Purification

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
10/10 PCR Cloning product AT 207.6 1.88 2.00

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
10/11 DNA Purification product AT(His) 10 0 1 0 1 D 4 0.4 24.6 40

Transformation

Yasuda
Sample CompetentCells Medium
10/10 Ligation BL21(DE3) CP

10/13

Miniprep

Yasuda
SampleConcentration/(µg/mL)260/280260/230
1. 10/13 T7-RBS (1)334.61.912.15
2. 10/13 T7-RBS (1)319.61.871.72

Electrophoresis

Yasuda
LaneSample
1-
2-
3-
41kb ladder
510/11 SAMmt-pSB1C3 (S)-(2)
610/11 SAMmt-pSB1C3 (S)-(3)
710/11 SAMmt-pSB1C3 (S)-(1)
810/11 SAMmt-pSB1C3 (S)-(4)
91kb ladder

Gel Extraction

Yasuda
LaneSample
1-
2-
310/11 AT-pSB1C3(X,P)-(2)
4-
5-
610/11 AT-His(X,P)
7-
81kb ladder
9-
1010/11 T7-RBS-His (S,P)-(2)
11-
12-
1310/11 T7-RBS-His (S,P)-(3)

Miniprep

Yasuda
SampleConcentration/(µg/mL)260/280260/230
1. 10/13 AT (X,P)(Gel Extraction Product)43.11.880.77
2. 10/13 AT-His (X,P)(Gel Extraction Product)34.61.960.37
3. 10/13 T7-RBS-His(2) (S,P)(Gel Extraction Product)57.71.841.29
4. 10/13 T7-RBS-His(3) (S,P)(Gel Extraction Product)37.51.811.10

Ligation

Yausda
Vector/(µL)Insert/(µL)Ligation high/(µL)Total/(µL)
1. 9/19 T7-RBS(S,P)(DNA purification product)0.810/13 AT(X,P)(Gel Extraction Product)2.02.85.6
2. 10/13 T7-RBS-His(S,P)-(2) (Gel Extraction Product)0.710/13 AT-His(X,P)(Gel Extraction Product)2.53.26.4
3. 10/13 T7-RBS-His(S,P)-(3) (Gel Extraction Product)0.810/13 AT-His(X,P)(Gel Extraction Product)1.92.75.4
4. 9/19 T7-RBS(S,P)(DNA purification product)0.8MilliQ2.02.85.6
5. 10/13 T7-RBS-His(S,P)-(2) (Gel Extraction Product)0.7MilliQ2.53.26.4
6. 10/13 T7-RBS-His(S,P)-(3) (Gel Extraction Product)0.8MilliQ1.92.75.4

Transformation

Yasuda
Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
10/13 Ligation Product (from 1 to 6)2DH5α1012CP
T7-RBS (1)(Miniprep product)2BL21(DE3)1012CP

Restriction Enzyme Digestion

Yasuda
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
10/13 T7-RBS (1)20--33B121.279.8120

Cloning

Yasuda
2x KAPA HiFi HotStart ReadyMix/(µL) Template DNA #1/(µL) Primers #2/(µL) MilliQ/(µL) Total/(µL)
12.5 pick up 0.75 11 25

(1)
#1: 10/10 SAMmt-pSB1C3(3) (After mutation, miniprep product)
#2: VF2, Rv SAMmt-His

(2)
#1: 10/10 SAMmt-pSB1C3(3) (After mutation, miniprep product)
#2: SAMmt(OE-A), Rv SAMmt-His

Predenature Denature Annealing Extension Last Extension Cooling
95°C 98°C 60°C 72°C 72°C 12°C
5min 20sec 15sec 1min15sec 5min Infinity

Denature-Annealing-Extension 30cycle

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
1. 10/14 SAMmt-pSB1C3(3)(PCR product)206.71.8811.17
2. 10/14 SAMmt-pSB1C3(3)(PCR product)187.91.8928.58

Restriction Enzyme Digestion

Yasuda
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
10/11 AT-pSB1C3 (2)(Miniprep product)5-0.5-0.5D20.211.820
10/14 SAMmt-His (1)(DNA purification product)10-1-1D20.423.640
10/14 SAMmt-His (2)(DNA purification product)10-1-1D20.423.640

10/14

Gel Extraction

Yasuda

(1)


LaneSample
11kb ladder
2-
310/14 AT(X,P)
4-
5-
610/14 SAMmt-His(2)(X,P)

(2)


LaneSample
1-
2-
310/13 T7-RBS(S,P)
41kb ladder
5-
610/11 T7-RBS-His(3) (S,P)

Transformation

Yasuda

10/15

Cloning

Yasuda

Sample/(µL)CompetentCells/(µL)Total/(µL)Medium
10/13 Ligation product (from 1 to 6)2DH5α1012CP
10/13 T7-RBS(1)2BL21(DE3)1012CP
Control (pBR322)2DH5α1012Amp
Control (pBR322)2DH5α1012Amp
Control (pBR322)2BL21(DE3)1012Amp
2x KAPA HiFi HotStart ReadyMix/(µL) 9/22 T7-RBS-dddD(After mutation, miniprep product)(3)/(µL) VF2/(µL) Rv dddD cloning for His/(µL) MilliQ/(µL) Total/(µL)
12.5 pick up 0.75 0.75 11 25
Predenature Denature Annealing Extension Last Extension Cooling
95°C 98°C 60°C 72°C 72°C 12°C
5min 20sec 15sec 1min15sec 5min Infinity

Denature-Annealing-Extension 30cycle

DNA Purification

Yasuda
SampleConcentration/(µg/mL)260/280260/230
10/14 AT(X,P)(Gel Extraction product)321.821.21
10/14 SAMmt-His(2)(X,P)(Gel Extraction product)701.781.01
10/14 T7-RBS(S,P)(Gel Extraction product)881.662.04
10/14 T7-RBS-His(3) (S,P)(Gel Extraction product)1561.541.10
10/15 dddD(After mutation, PCR product)3461.851.44

Restriction Enzyme Digestion

yasuda
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
10/15 dddD(After mutation, DNA purification product)5.8-1-1D40.427.840

Liquid Culture

Yasuda
SampleMedium
9/8 T'-RBS-pSB1C3(1)CP
REDOX-pSB1C3CP
10/3 AT-pSB1C3CP
T7-RBS-His(2)CP

10/16

Gel Extraction/Electrophoresis

Yasuda
Lane Sample
1 1kbpGeneRuler
2 dddD(His)X,P

Miniprep

Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
T7RBS-pSB1C3 (1) 10/16 Miniprep product 471 1.90 1.85
T7RBS-pSB1C3 (2) 10/16 Miniprep product A 380 1.92 2.03
T7RBS-pSB1C3 (2) 10/16 Miniprep product B 372 1.92 1.64
AC-pSB1C3(2) 10/16 Miniprep product 473 1.90 1.68
REDOX-pSB1C3 10/16 Miniprep product 530 1.93 1.81
dddD(His)X,P 10/16 gel extraction product 55 1.40 1.36
QpSB1C3 10/16 Miniprep product 276 1.86 2.01
PLQ-4A5 10/16 Miniprep product 370 1.87 2.07

Ligation

Honda

Toyobo Ligationhigh

Vector/(µL) Insert/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
10/14 T7-RBS S,P 2 10/14 AT X,P 2 2xligationhigh 4 - 8
10/14 T7-RBS S,P 2 10/14 AT X,P - 2xligationhigh 4 2 8
10/14 T7-RBS-His S,P 1 10/13 AT-His X,P 2 2xligationhigh 4 1 8
10/14 T7-RBS-His S,P 1 10/13 AT-His X,P - 2xligationhigh 4 3 8

Promega

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
10/14 T7-RBS S,P 2 10/14 AT X,P 2 2xRapidLigationBuffer 5 T4Ligase 1 - 10
10/14 T7-RBS S,P 2 10/14 AT X,P - 2xRapidLigationBuffer 5 T4Ligase 1 2 10
10/14 T7-RBS-His S,P 1 10/13 AT-His X,P 2 2xRapidLigationBuffer 5 T4Ligase 1 1 10
10/14 T7-RBS-His S,P 2 10/14 SAM-His X,P 2 2xRapidLigationBuffer 5 T4Ligase 1 - 10
10/14 T7-RBS-His S,P 2 REDOX-His 2 2xRapidLigationBuffer 5 T4Ligase 1 - 10
10/14 T7-RBS-His S,P 2 REDOX-His - 2xRapidLigationBuffer 5 T4Ligase 1 2 10

NewIngland BioLabs

Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
10/14 T7-RBS-His S,P 2 10/14 SAM-His X,P 2 10xLigationBuffer 0.5 T4 Ligase 0.5 - 5
10/14 T7-RBS-His S,P 2 REDOX-His 2 10xLigationBuffer 0.5 T4 Ligase 0.5 - 5
10/14 T7-RBS-His S,P 2 REDOX-His - 10xLigationBuffer 0.5 T4 Ligase 0.5 2 5

Transformation

Honda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
Toyobo T7-AT 2 BL21 10 12 CP
Toyobo T7-AT control 2 BL21 10 12 CP
Toyobo T7-AT-His 2 BL21 10 12 CP
Toyobo T7-AT-His control 2 BL21 10 12 CP
10/13 Miniprep product T7-RBS(1)B 9 BL21 90 99 CP
Toyobo T7-AT 4 DH5α 20 24 CP
Toyobo T7-AT control 4 DH5α 20 24 CP
Toyobo T7-AT-His 4 DH5α 20 24 CP
Toyobo T7-AT-His control 4 DH5α 20 24 CP
Promega T7-AT 5 DH5α 30 35 CP
Promega T7-AT control 5 DH5α 30 35 CP
Promega T7-AT-His 5 DH5α 30 35 CP
Promega T7-SAM-His 5 DH5α 30 35 CP
Promega T7-REDOX-His 5 DH5α 30 35 CP
Promega T7-REDOX-His control 5 DH5α 30 35 CP
NewIngland T7-SAM-His 5 DH5α 30 35 CP
NewIngland T7-REDOX-His 5 DH5α 30 35 CP
NewIngland T7-REDOX-His control 5 DH5α 30 35 CP

Retrieved from "http://2014.igem.org/Team:Kyoto/Notebook/DMS_Synthesis"