Template:Kyoto/Notebook/DMS/23

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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
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

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 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

3/17

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

　　　　　

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

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

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