Template:Kyoto/Notebook/DMS

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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

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 Kabber(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

Transformation

Yasuda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
2/12 J23118(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) 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 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 2/12 I712074(Ak) 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 2100bp 6 - - 0.5 0.5 H 3 3 17 30
9/8 J23100 2100bp 0.3 - - - - H 1 1 7.7 10
9/28 RBS-GFP-DT 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 G23100 S,P
3 9/8 G23100 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 9/2 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 2100bp 4.8 3/4 RBS-GFP-DT X,P 900bp 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 Tag/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
65 2.6 2.6 59.8
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 24sec

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 J712074 (1)
6 3/5 J712074 (2)
7 3/5 J712074 (3)
8 3/5 J712074 (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) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
50 2 2 46 100
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 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) VF2/(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 (1)
5 3/10 pSB1C3 (2)
6 3/10 DT (1)
7 3/10 DT (2)
8 3/10 T7 (1)
9 3/10 T7 (2)
10 old Quick Taq control (9)
11 new Quick Taq control (10)
12 1kbp ladder

Colony PCR

Kojima

Master Mix

Quick Tag/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
17.5 0.7 0.7 16.1(MilliQ) 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 Tag/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
17.5 0.7 0.7 16.1(MilliQ) 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

3/17

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

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

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) F/(µL) R/(µL) HiFi Mix/(µL) MilliQ/(µL) Total/(µL)
1 0.75 0.75 12.5 10 25
R.pomeroyi Genome/(µL) F/(µL) R/(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
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/(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 dddD
Sample Concentration/(µg/mL) 260/280 260/230
dddD 63.7 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
pSB1C3 3/24 (4) 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 30 dddD 30 2x Rapid Ligation Buffer 5 T4Ligase 3 - 10

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 Sequence primer (1)
2 Sequence primer (2)
3 Sequence primer (3)
4 Sequence primer (4)
5 Sequence primer (5)
6 Sequence primer (6)
7 Sequence primer VF2
8 F pre-dddD PCR product 62
9 Blank

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) VF2/(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
2min 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

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

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

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
105 1.30 0.85
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 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 0.81 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 2h20min

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 KARA 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 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 DH5a 40 50 SOC
8/29 ligation product control 10 DH5a 40 50 SOC
pSB1C3 control 1 DH5a 20 21 SOC

9/1

Colony PCR

Honda

Master Mix

Quick Tag/(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 Tag/(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 Extention Cooling
95°C 98°C 65°C 72°C 72°C 4°C
5min 20sec 15sec 2min10sec 5min long time

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 Extention Cooling
95°C 98°C 65°C 72°C 72°C 4°C
5min 20sec 15sec 2min10sec 5min long time

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,S)
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 Extention Cooling
98°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min long time

Denature-Annealing-Extension 25cycle

(2)(3)
Predenature Denature Annealing Extension final Extention Cooling
98°C 98°C 60°C 72°C 72°C 4°C
5min 20sec 15sec 70sec 5min long time

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)7.81-1-3-17.230
I732095 8/22 miniprep (2)(255ng/µL)0.4----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)7.81-1-3-17.230
I732095 8/22 miniprep (2)(255ng/µL)0.4----1-8.610
I732095 8/22 miniprep (1)(430ng/µL)4.61-1-3-20.430

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

ゲノムクローニング@

Honda

Electrophoresis

Honda (1)AT
LaneSample
1-
2-
3-
41kb ladder
5
6
7
8
9
10
11
12
131kb ladder
14-
15-
16-
17-
<
(2)SAMmt
LaneSample
1-
2-
3-
41kb ladder
5
6
7
8
9
10
11-
12-
13-
14-
15-
16-
171kb ladder
<
(3)DiDECARB
LaneSample
1-
2-
3-
41kb ladder
5
6
7
8
9
10
11
12
131kb ladder
14-
15-
16-
17-

9/8

ゲノムクローニング@

Honda and Yamaura

PCR@

Honda and Yamaura

Electrophoresis

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

カラム精製

Matsumoto

Restriction Enzyme Digestion

Matsumoto
Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
サンプル名(サンプル)(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)30
control(サンプル)(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)10

PCR

Yasuda

パーツ起こし

Honda

9/9

カラム精製

Honda

Ligation

Li (1)Sample (2)Control
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
pSB1C34.2dddD3.89T4 Ligase1-18
pSB1C34.2--9T4 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
サンプル名培地

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
95°C 98°C 64°C 72°C72°C12°C
5min 20sec 15sec 50sec 5min long time

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@

Murata

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/DNA Purification

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 Tag/(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

Electrophoresis

Honda
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 Extention(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, AT 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

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