Template:Kyoto/Notebook/DMS

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<h4>Ligation</h4>
<h4>Ligation</h4>
<span class="kyoto-author">Yasuda</span>
<span class="kyoto-author">Yasuda</span>

Revision as of 15:49, 4 October 2014

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-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

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
3min 20sec 15sec 150sec 1sec long time

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 long time

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 long time

Denature-Annealing-Extension 25cycle

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)
pSB1C34.2dddD3.8(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
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

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 60°C 72°C 72°C 4°C
5min 20sec 15sec 90sec 5min long time

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/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, 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)@

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 Extention Cooling
98°C 98°C 55°C 72°C 72°C 12°C
5min 20sec 15sec 2min10sec 5min long time

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 Last Extention Cooling
98°C 98°C 55°C 72°C 72°C 12°C
5min 10sec 15sec 25sec 5min long time

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)

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 Extention Cooling
98°C 98°C 55°C 72°C 72°C 12°C
2min 10sec 10sec 1min 5min long time

Denature-Annealing-Extension 25cycle

9/12

Electrophoresis

Li
LaneSample
11kb ladder
29/11 OE-PCR 2nd (1)
39/11 OE-PCR 2nd (2)
49/11 OE-PCR 2nd (3)
59/11 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)2DH5&alfa;1012SOD@
9/11 DiDECARB (ligation product)2DH5&alfa;1012SOD
9/11 DECARB (ligation product)2DH5&alfa;1012SOD
9/11 REDOX (ligation product)2DH5&alfa;1012SOD

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)SOD@

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 12°C
5min 20sec 15sec 90sec 5min long time

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 long time

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 65°C 72°C 72°C 12°C
5min 20sec 15sec 45sec 5min long time

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 111.5
Vector/(µL)Insert/(µL)Buffer/(µL)Ligase/(µL)MilliQ/(µL)Total/(µL)
YC plac111AT-His cloning0.5(2x)Buffer5T4 Ligase13.510
YC plac111AT0.5(2x)Buffer5T4 Ligase13.510
psB1C3 (E,P)SAMmt (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

Electrophoresis@

Tatsui and Li
LaneSample
1YCplac111 SAMmt(1)
2YCplac111 SAMmt(2)
3YCplac111 SAMmt(3)
4YCplac111 SAMmt(4)
5YCplac111 SAMmt(5)
6YCplac111 SAMmt(6)
7YCplac111 SAMmt(7)
8YCplac111 SAMmt(8)
91kb ladder
10YCplac111 REDOX(1)
11YCplac111 REDOX(2)
12YCplac111 REDOX(3)
13YCplac111 REDOX(4)
14YCplac111 REDOX(5)
15YCplac111 REDOX(6)
16YCplac111 REDOX(7)

Liquid Culture@

Yamauchi AmpCPCP
SampleMedium
9/5 Pcon-psB1C3-(3)CP
9/5 Pcon-psB1C3-(3)CP
9/11 SAMmt-(3)
9/8 T7-RBS-(1)
9/8 T7-RBS-(1)

Colony PCR

Honda

Master Mix

Quick Tag/(µL) M13 Fw/(µL) M13 Rv/(µL) MilliQ/(µL) Total/(µL)
35 1.4 1.4 32.2 70

1. REDOX-YCplac111-(1)
2. REDOX-YCplac111-(2)
3. REDOX-YCplac111-(3)
4. REDOX-YCplac111-(4)
5. REDOX-YCplac111-(5)
6. REDOX-YCplac111-(6)
7. REDOX-YCplac111-(7)

Predenature Denature Annealing Extension Last Extention Cooling
94°C 94°C 55°C 68°C 68°C 12°C
2min 30sec 30sec 1min 1sec long time

Denature-Annealing-Extension 25cycle

Electrophoresis@

Honda
LaneSample
1
2
3
4
5
6
7
8
91kb ladder
10REDOX-YCplac111-(1)
11REDOX-YCplac111-(2)
12REDOX-YCplac111-(3)
13REDOX-YCplac111-(4)
14REDOX-YCplac111-(5)
15REDOX-YCplac111-(6)
16REDOX-YCplac111-(7)
17REDOX-YCplac111-(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))

Miniprep/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
94°C 94°C 55°C 68°C
2min 30sec 30sec 160sec

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 Tag/(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(µ)-psB1C3(X,P)-(1)
4-
51kb ladder
6-
79/14 dddD(µ)-psB1C3(X,P)-(2)
8-
91kb ladder
10-
119/14 dddD(µ)-psB1C3(X,P)-(3)

DNA Purification

Tatsui
SampleConcentration/(µg/mL)260/280260/230
9/14 dddD(µ)-psB1C3(X,P)-(1)1.7 (x50)1.370.33
9/14 dddD(µ)-psB1C3(X,P)-(2)1.6 (x50)1.410.36
9/14 dddD(µ)-psB1C3(X,P)-(3)2.2 (x50)1.230.42
T7-RBS-psB1C3(S cut)2.0 (x50)2.110.34

Restriction Enzyme Digestion@

Sample/(µL)EcoR1/(µL)Xba1/(µL)Spe1/(µL)Pst1/(µL)Buffer/(µL)BSA/(µL)MilliQ/(µL)Total/(µL)
9/13 T7-RBS-psB1C37(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)30
9/11 REDOX (DNA purification product)10(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)30
9/13 Pcon-psB1C325(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)30
9/15 T7-RBS-psB1C325(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)30

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
サンプル名(サンプル)コンピ名(コンピ)(Total)培地
サンプル名(サンプル)コンピ名(コンピ)(Total)培地
サンプル名(サンプル)コンピ名(コンピ)(Total)培地
サンプル名(サンプル)コンピ名(コンピ)(Total)培地

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(µ)(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-YCplac1112DH5&alfa;1012SOD
DiDECARB-YCplac1112DH5&alfa;1012SOD
dddD(µ)-T7-RBS-psB1C32DH5&alfa;1012SOD
SAMmt-psB1C32DH5&alfa;1012SOD
REDOX-psB1C32DH5&alfa;1012SOD
AT-T7-RBS-psB1C32DH5&alfa;1012SOD

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 Extention Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min long time

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 Extention Cooling
96°C 96°C 50°C 60°C 4°C 4°C
2min 10sec 5sec 4min 1min long time

Denature-Annealing-Extension 30cycle

Dephosphorylation@

Honda
Sample/(µL)
1.(サンプル)
2.(サンプル)
3.9/15 T7-RBS-psB1C3(P)(サンプル)
4.9/15 T7-RBS-psB1C3(S,P)(サンプル)
5.9/15 T7-RBS-psB1C3(S,P)(サンプル)
6.9/15 T7-RBS-psB1C3(S,P)(サンプル)
7.9/15 T7-RBS-psB1C3(S,P)(サンプル)

Colony PCR

Yasuda

Master Mix

Quick Tag/(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(µ)(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(µ)-T7-RBS-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