|
|
| Line 2,347: |
Line 2,347: |
| | <tr><td>7</td><td>1kb ladder</td> | | <tr><td>7</td><td>1kb ladder</td> |
| | </table> | | </table> |
| | + | <img src="https://static.igem.org/mediawiki/2014/a/a1/Kyoto-dms-0904_electrophoresis_No1.jpg"> |
| | | | |
| | <!--"ゲノムを水？で戻す"を省略--> | | <!--"ゲノムを水？で戻す"を省略--> |
| Line 2,505: |
Line 2,506: |
| | <tr><td>6</td><td>1kb ladder</td> | | <tr><td>6</td><td>1kb ladder</td> |
| | </table> | | </table> |
| - | | + | <img src="https://static.igem.org/mediawiki/2014/d/db/Kyoto-dms-0904_cloning_electrophoresis_No1.jpg"> |
| | | | |
| | <h4>Restriction Enzyme Digestion</h4> | | <h4>Restriction Enzyme Digestion</h4> |
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
| 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 |
Electrophoresis
Nakamura
| 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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/1 dddD+pSB1C3-1 |
| 3 | 9/1 dddD+pSB1C3-2 |
| 4 | 9/1 dddD+pSB1C3-3 |
| 5 | 9/1 pSB4A5-1 |
| 6 | 9/1 pSB4A5-2 |
| 7 | control |
| 8 | 1kb 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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/1 dddD+pSB1C3-1 |
| 3 | 9/1 dddD+pSB1C3-2 |
| 4 | 9/1 dddD+pSB1C3-3 |
| 5 | 9/1 pSB4A5-1 |
| 6 | 9/1 pSB4A5-2 |
| 7 | control |
| 8 | 1kb ladder |
9/2
Electrophoresis
Mastumoto
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 8/29 dddD(DNA purification product)1µL+MilliQ 5µL+looding dye(x6)1µL |
| 3 | control |
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) | 6 | 1 | - | 1 | - | - | 3 | - | 19 | 30 |
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
| Lane | Sample |
|---|
| 2 | dddD (E,S) 2544bp. |
| 4 | dddD (E,S) |
| 6 | 1kb ladder |
DNA Purification
Matsumoto
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| Pre-dddD-suf | 685 | 1.66 | 2.13 |
Ligation
Honda, Li and Yamauchi
(1)insert: dddD
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| pSB1C3 | 1.5 | dddD | 6.3 | 2x Buffer | 8.8 | T4 Ligase | 1 | - | 17.6 |
(2)control
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| pSB1C3 | 1.5 | dddD | - | 2x Buffer | 8.8 | T4 Ligase | 1 | 6.3 | 17.6 |
Transformation
Yamauchi
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| 9/3 Ligation product (dddD) | 3 | DH5α | 15 | 18 | SOC |
| 9/3 Ligation product (control) | 2 | DH5α | 10 | 12 | SOC |
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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/3 Ligation dddD + pSB1C3 |
| 3 | 8/20 Ligation dddD + pSB1C3 |
| 4 | 8/1 Ligation dddD + pSB1C3 |
| 5 | 9/3 dddD (Gel extraction product)(E,S) |
| 6 | 9/3 dddD (DNA purification product) |
| 7 | 1kb 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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | AT cloning(1) |
| 3 | SAMmt cloning(2) |
| 4 | DECARB cloning(3) |
| 5 | DiDECARB cloning(4) |
| 6 | 1kb 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) | 7.8 | 1 | - | 1 | - | H(x10) | 3 | - | 17.2 | 30 |
| I732095 8/22 miniprep (2)(255ng/µL) | 0.4 | - | - | - | - | H(x10) | 1 | - | 8.6 | 10 |
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) | 7.8 | 1 | - | 1 | - | H(x10) | 3 | - | 17.2 | 30 |
| I732095 8/22 miniprep (2)(255ng/µL) | 0.4 | - | - | - | - | H(x10) | 1 | - | 8.6 | 10 |
| I732095 8/22 miniprep (1)(430ng/µL) | 4.6 | 1 | - | 1 | - | H(x10) | 3 | - | 20.4 | 30 |
9/5
Gel Extraction
Honda and Yamauchi
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/4 I732095(E,S)-1 |
| 3 | - |
| 4 | 9/4 I732095(E,S)-1 |
| 5 | - |
| 6 | 9/4 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
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | - |
| 4 | 1kb 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 |
| 13 | 1kb ladder |
| 14 | - |
| 15 | - |
| 16 | - |
| 17 | - |
(2)SAMmt
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | - |
| 4 | 1kb 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 |
| 17 | 1kb ladder |
(3)DiDECARB
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | - |
| 4 | 1kb 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 |
| 13 | 1kb ladder |
| 14 | - |
| 15 | - |
| 16 | - |
| 17 | - |
Gel Extraction
Murata
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/4 I732045(E,S) |
| 3 | 9/4 I732045(E,S) |
| 4 | - |
| 5 | - |
| 6 | 9/4 I732045(E,S) |
| 7 | 9/4 I732045(E,S) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | 9/4 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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | PCR product (AT) |
| 3 | PCR product (DIDECARB) |
| 4 | PCR product (dddD) |
| 5 | PCR product (dddD) |
| 6 | 1kb ladder |
DNA Purification
Matsumoto
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/8 AT | 55 | 1.37 | 1.14 |
| 9/8 DiDECARB | 95 | 1.69 | 3.04 |
| 9/8 dddD | 145 | 1.52 | 1.78 |
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) | 13.8 | 1 | - | 1 | - | H | 3 | - | 11.2 | 30 |
| control | 0.7 | - | - | - | - | H | 1 | - | 8.3 | 10 |
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
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| pSB1C3 | 4.2 | dddD | 3.8 | (2x)Buffer | 9 | T4 Ligase | 1 | - | 18 |
(2)Control
| pSB1C3 | 4.2 | - | - | (2x)Buffer | 9 | T4 Ligase | 1 | 3.8 | 18 |
Transformation
Li
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| 9/9 dddD+pSB1C3 ligation product | 5 | DH5α | 50 | 55 | SOC |
| 9/9 dddD+pSB1C3 ligation product control | 3 | DH5α | 30 | 33 | SOC |
Liquid Culture
Honda
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°C |
72°C |
12°C |
| 5min |
20sec |
15sec |
50sec |
5min |
long time |
Denature-Annealing-Extension 25cycle
Gel Extraction
Murata
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/8 SAMmt PCR product (5µL) |
| 3 | - |
| 4 | 9/8 SAMmt PCR product (10µL) |
| 5 | 9/8 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
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | 1kb ladder (5µL) |
| 4 | 9/9 REDOX PCR product (2µL) |
| 5 | 9/9 SAMmt PCR product (2µL) |
| 6 | 1kb ladder (5µL) |
| 7 | - |
| 8 | - |
Miniprep/DNA Purification
Murata
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| T7-RBS | 290 | 1.90 | 1.83 |
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
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | - |
| 4 | 1kb ladder |
| 5 | SAMmt (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
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | - |
| 4 | 1kb ladder |
| 5 | 9/9 dddD+psb1c3 (1) |
| 6 | 9/9 dddD+psb1c3 (2) |
| 7 | 9/9 dddD+psb1c3 (3) |
| 8 | 9/9 dddD+psb1c3 (4) |
| 9 | 1kb ladder |
| 10 | 9/9 dddD+psb1c3 (5) |
| 11 | 9/9 dddD+psb1c3 (6) |
| 12 | 9/9 dddD+psb1c3 (control) |
| 13 | control |
| 14 | 1kb ladder |
| 15 | - |
| 16 | - |
| 17 | - |
Electrophoresis
Yasuda
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | 1kb ladder |
| 4 | REDOX 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 |
| Number | AnnealingTemperature |
|---|
| 1 | 65.0 °C |
| 2 | 64.3 °C |
| 3 | 63.0 °C |
| 4 | 61.1 °C |
| 5 | 58.8 °C |
| 6 | 56.9 °C |
| 7 | 55.7 °C |
| 8 | 55.0 °C |
Gel Extraction
Yasuda
(1)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/10 OE-PCR(1) |
| 3 | 9/10 OE-PCR(1) |
| 4 | - |
| 5 | 9/10 OE-PCR(2) |
| 6 | - |
| 7 | 9/10 OE-PCR(2) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | 9/10 OE-PCR(3) |
| 11 | 9/10 OE-PCR(3) |
(2)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/10 OE-PCR(4) |
| 3 | 9/10 OE-PCR(4) |
| 4 | - |
| 5 | 9/10 OE-PCR(5) |
| 6 | 9/10 OE-PCR(5) |
| 7 | 1kb ladder |
(3)
| Lane | Sample |
|---|
| 1 | - |
| 2 | 1kb ladder |
| 3 | 9/10 OE-PCR(6) |
| 4 | 9/10 OE-PCR(6) |
(4)
| Lane | Sample |
|---|
| 1 | - |
| 2 | 1kb ladder |
| 3 | 9/10 OE-PCR(7) |
| 4 | 9/10 OE-PCR(7) |
(5)
| Lane | Sample |
|---|
| 1 | - |
| 2 | 1kb ladder |
| 3 | 9/10 OE-PCR(8) |
| 4 | 9/10 OE-PCR(8) |
(6)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | 9/10 OE-PCR(9) |
| 4 | 9/10 OE-PCR(9) |
| 5 | 9/10 OE-PCR(10) |
| 6 | 9/10 OE-PCR(10) |
Ligation
Tatsui and Murata
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| YC plac111 (SmaI cut) | 1 | 9/8 dddD(DNA purification product) | 0.6 | (2x)Buffer | 5 | T4 Ligase | 1 | 2.4 | 10 |
Liquid Culture
Honda
| Sample | Medium |
|---|
| 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-RBS | 6.9 | - | - | 1 | 1 | H | 3 | - | 18.1 | 30 |
| control | 1 | - | - | - | - | H | 1 | - | 8 | 10 |
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) |
|---|
| YCplac111 | 1 | dddD | 1 | (2x)Buffer | 5 | T4 Ligase | 1 | 2 | 10 |
9/11
Miniprep
Murata
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/10 dddD (liquid culture product) | 165 | 1.88 | 2.83 |
DNA Purification
Matsumoto
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/10 REDOX(5)(6) (genome cloning product) | 55 | 1.56 | 1.32 |
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
| Lane | Sample |
|---|
| 1 | - |
| 2 | 1kb ladder |
| 3 | 9/10 OE-PCR-(1) |
| 4 | 9/10 OE-PCR-(2) |
| 5 | 9/10 OE-PCR-(3) |
| 6 | 9/10 OE-PCR-(3') |
| 7 | 1kb 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 |
| 1 | LB Amp. 9/10 mamB(1) |
| 2 | LB Amp. 9/10 mamB(2) |
| 3 | LB Amp. 9/10 mamB(3) |
| 4 | LB Amp. 9/10 mamB(4) |
| 5 | LB Amp. 9/10 mamB(5) |
| 6 | LB Amp. 9/10 mamB(6) |
| 7 | LB Amp. 9/10 mamB(7) |
| 8 | LB Amp. 9/10 mamB(8) |
(2)
| Number |
Sample |
| 1 | LB Amp. 9/10 mamL(1) |
| 2 | LB Amp. 9/10 mamL(2) |
| 3 | LB Amp. 9/10 mamL(3) |
| 4 | LB Amp. 9/10 mamL(4) |
| 5 | LB Amp. 9/10 mamL(5) |
| 6 | LB Amp. 9/10 mamL(6) |
| 7 | LB Amp. 9/10 mamL(7) |
| 8 | LB Amp. 9/10 mamL(8) |
(3)
| Number |
Sample |
| 1 | LB Amp. 9/10 mamQ(1) |
| 2 | LB Amp. 9/10 mamQ(2) |
| 3 | LB Amp. 9/10 mamQ(3) |
| 4 | LB Amp. 9/10 mamQ(4) |
| 5 | LB Amp. 9/10 mamQ(5) |
| 6 | LB Amp. 9/10 mamQ(6) |
| 7 | LB Amp. 9/10 mamQ(7) |
| 8 | LB Amp. 9/10 mamQ(8) |
(4)
| Number |
Sample |
| 1 | LB Amp. 9/10 dddD(1) |
| 2 | LB Amp. 9/10 dddD(2) |
| 3 | LB Amp. 9/10 dddD(3) |
| 4 | LB Amp. 9/10 dddD(4) |
| 5 | LB Amp. 9/10 dddD(5) |
| 6 | LB Amp. 9/10 dddD(6) |
| 7 | LB Amp. 9/10 dddD(7) |
| 8 | LB 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
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| dddD, T7-RBS-His | - | - | - |
Gel Extraction
Honda
(1)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/11 OE-PCR-(1) |
| 3 | 9/11 OE-PCR-(1) |
| 4 | - |
| 5 | - |
| 6 | 9/11 OE-PCR-(2) |
| 7 | 9/11 OE-PCR-(2) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | 9/11 OE-PCR-(3) |
| 11 | 9/11 OE-PCR-(3) |
(2)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/11 OE-PCR-(6) |
| 3 | 9/11 OE-PCR-(6) |
| 4 | - |
| 5 | - |
| 6 | 9/11 OE-PCR-(8) |
| 7 | 9/11 OE-PCR-(8) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | 9/11 OE-PCR-(10) |
| 11 | 9/11 OE-PCR-(10) |
(3)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/11 OE-PCR-(12) |
| 3 | 9/11 OE-PCR-(12) |
| 4 | 9/11 OE-PCR (REDOX 2nd PCR) |
| 5 | - |
| 6 | 9/11 OE-PCR-(14) |
| 7 | 9/11 OE-PCR-(14) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | 9/11 OE-PCR-(16) |
| 11 | 9/11 OE-PCR-(16) |
DNA Purification
Yamauchi
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/10 OE-PCR (1) SAMmt | 975 | 1.06 | 1.08 |
| 9/10 OE-PCR (2) DECARB | 420 | 1.67 | 2.19 |
| 9/20 OE-PCR (3) DiDECARB | 175 | 1.66 | 2.45 |
| 9/10 OE-PCR (3') DiDECARB | 215 | 1.57 | 1.82 |
| 9/11 Mutation PCR dddD | 275 | 1.61 | 1.92 |
| 9/11 Mutation PCR T7 | 140 | 1.49 | 1.78 |
| Control C | 340 | 1.62 | 1.47 |
Ligation
Tatsui and Yamauchi
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| YC plac111 (SmaI cut) | 1 | 9/11 SAMmt (DNA purification product) | 0.5 | (2x) Buufer | 5 | T4 Ligase | 1 | 2.5 | 9 |
| YC plac111 (SmaI cut) | 1 | 9/11 DiDECARB (DNA purification product) | 1 | (2x) Buufer | 5 | T4 Ligase | 1 | 2 | 9 |
| YC plac111 (SmaI cut) | 1 | 9/11 DECARB (DNA purification product) | 1 | (2x) Buufer | 5 | T4 Ligase | 1 | 2 | 9 |
| YC plac111 (SmaI cut) | 1 | 9/11 REDOX (DNA purification product) | 2 | (2x) Buufer | 5 | T4 Ligase | 1 | 1 | 9 |
| 9/10 Pcon-X(PstI cut, gel extraction product) | 1.5 | mamL(X.P) | 2 | (2x)Buffer | 5 | T4 Ligase | 1 | - | 9.5 |
| 9/10 Pcon-X(PstI cut, gel extraction product) | 1.5 | mamQ(X.P) | 2 | (2x)Buffer | 5 | T4 Ligase | 1 | - | 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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/11 OE-PCR 2nd (1) |
| 3 | 9/11 OE-PCR 2nd (2) |
| 4 | 9/11 OE-PCR 2nd (3) |
| 5 | 9/11 OE-PCR 2nd (4) |
DNA Purification
Li
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/11 OE-PCR 2nd (1) | 220 | 1.65 | 1.96 |
| 9/11 OE-PCR 2nd (2) | 240 | 1.65 | 2.10 |
| 9/11 OE-PCR 2nd (3) | 225 | 1.68 | 2.03 |
| 9/11 OE-PCR 2nd (4) | 160 | 1.64 | 1.91 |
Transformation
Yamauchi
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| 9/11 SAMmt (ligation product) | 2 | DH5&alfa; | 10 | 12 | SOD@ |
| 9/11 DiDECARB (ligation product) | 2 | DH5&alfa; | 10 | 12 | SOD |
| 9/11 DECARB (ligation product) | 2 | DH5&alfa; | 10 | 12 | SOD |
| 9/11 REDOX (ligation product) | 2 | DH5&alfa; | 10 | 12 | SOD |
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
| Sample | Medium |
|---|
| 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 SAMmt | 2 | 1 | - | - | 1 | H | 3 | - | 23 | 30 |
| 9/10 OE-PCR DECARB | 4.8 | - | 1 | 1 | - | M | 3 | 3 | 17.2 | 30 |
| 9/10 OE-PCR DiDECARB | 12.4 | - | 1 | 1 | - | M | 3 | 3 | 10.6 | 30 |
| 9/10 OE-PCR T7-RBS | 6.9 | - | - | 1 | 1 | H | 3 | - | 18.1 | 30 |
| Control | 2.3 | - | - | - | - | H | 1 | - | 5 | 9.9 |
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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | Control(9/11 T7-RBS) |
| 3 | - |
| 4 | 9//11 T7-RBS(S,P) |
| 5 | - |
| 6 | 1kb 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
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| dddD-psB1C3 (Liquid culture product) | 370 | 1.75 | 2.48 |
DNA Purification
Tatsui
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 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) | 9 | 1 | - | - | 1 | H | 3 | - | 16 | 30 |
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 | - | 1 | M | 3 | 3 | 13.3 | 30 |
| Control | 0.4 | - | - | - | - | M | 3 | 0 | 6.6 | 10 |
| 8/2 Pcon | 20 | 1 | - | 1 | - | H | 3 | 3 | 2 | 30 |
| Control | pick up | - | - | - | - | H | 3 | 0 | 7 | 10 |
Ligation
Honda
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| YC plac111 | 1 | AT-His cloning | 0.5 | (2x)Buffer | 5 | T4 Ligase | 1 | 3.5 | 10 |
| YC plac111 | 1 | AT | 0.5 | (2x)Buffer | 5 | T4 Ligase | 1 | 3.5 | 10 |
| psB1C3 (E,P) | 1.5 | SAMmt (E,P) | 1 | (2x)Buffer | 5 | T4 Ligase | 1 | 2.5 | 10 |
DNA Purification
Tatsui
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/11 DECARB (Restriction Enzyme Digestion product) | 40 | 1.74 | 1.95 |
| 9/11 DiDECARB (Restriction Enzyme Digestion product) | 30 | 2.00 | 1.70 |
| 9/11 SAMmt (Restriction Enzyme Digestion product) | 20 | 3.54 | 2.33 |
| 9/11 T7-RBS | 60 | 1.81 | 0.27 |
| 9/10 T7-RBS | 50 | 1.65 | 1.77 |
Electrophoresis
| Lane | Sample |
|---|
| 1 | - |
| 2 | - |
| 3 | - |
| 4 | 1kb ladder |
| 5 | 9/11 E-PCR 2nd (1) |
| 6 | 9/11 Control (PCR product) (1) |
| 7 | 9/11 E-PCR 2nd (2) |
| 8 | 9/11 Control (PCR product) (2) |
| 9 | 9/11 E-PCR 2nd (3) |
| 10 | 9/11 Control (PCR product) (3) |
| 11 | 9/11 E-PCR 2nd (4) |
| 12 | 9/11 Control (PCR product) (4) |
| 13 | 1kb ladder |
| 14 | - |
| 15 | - |
| 16 | - |
Transformation
Honda
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| AT-His-YCplac111 | 1 | DH5α | 10 | 11 | SOD |
| AT-YCplac111 | 1 | DH5α | 10 | 11 | SOD |
| SAMmt(E,P)-psB1C3(E,P) | 1 | DH5α | 10 | 11 | SOD |
Transformation
Yasuda
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| 9/11 dddD (DNA purification product) | 1 | DH5α | 10 | 11 | SOD |
| 9/11 T7-His (DNA purification product) | 1 | DH5α | 10 | 11 | SOD |
Colony PCR@
Tatsui and Li
Electrophoresis@
Tatsui and Li
| Lane | Sample |
|---|
| 1 | YCplac111 SAMmt(1) |
| 2 | YCplac111 SAMmt(2) |
| 3 | YCplac111 SAMmt(3) |
| 4 | YCplac111 SAMmt(4) |
| 5 | YCplac111 SAMmt(5) |
| 6 | YCplac111 SAMmt(6) |
| 7 | YCplac111 SAMmt(7) |
| 8 | YCplac111 SAMmt(8) |
| 9 | 1kb ladder |
| 10 | YCplac111 REDOX(1) |
| 11 | YCplac111 REDOX(2) |
| 12 | YCplac111 REDOX(3) |
| 13 | YCplac111 REDOX(4) |
| 14 | YCplac111 REDOX(5) |
| 15 | YCplac111 REDOX(6) |
| 16 | YCplac111 REDOX(7) |
Liquid Culture@
Yamauchi
| Sample | Medium |
|---|
| 9/5 Pcon-psB1C3-(3) | CP |
| 9/5 Pcon-psB1C3-(3) | CP |
| 9/11 SAMmt-(3) | | Amp
| 9/8 T7-RBS-(1) | | CP
| 9/8 T7-RBS-(1) | | CP
Colony PCR
Honda
Master Mix
| Quick 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
| Lane | Sample |
|---|
| 1 | |
| 2 | |
| 3 | |
| 4 | |
| 5 | |
| 6 | |
| 7 | |
| 8 | |
| 9 | 1kb ladder |
| 10 | REDOX-YCplac111-(1) |
| 11 | REDOX-YCplac111-(2) |
| 12 | REDOX-YCplac111-(3) |
| 13 | REDOX-YCplac111-(4) |
| 14 | REDOX-YCplac111-(5) |
| 15 | REDOX-YCplac111-(6) |
| 16 | REDOX-YCplac111-(7) |
| 17 | REDOX-YCplac111-(8) |
9/13
Miniprep
Honda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/12 T7-RBS-psB1C3 (liquid culture product) | 95 | 2.02 | 2.72 |
| 9/12 SAMmt-YCplac111 (liquid culture product) | 110 | 1.70 | 1.15 |
Gel Extraction
Honda and Yasuda
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | - |
| 4 | psB1C3-Pcon(X,S) |
| 5 | - |
| 6 | Control (psB1C3-Pcon(X,S)) |
Miniprep/DNA Purification
Yasuda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/12 AT (E,P) | 2.3 | 1.33 | 1.11 |
| 9/12 AT-His(X,P) | 2.0 | 1.33 | 0.86 |
Ligation
Honda
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| 9/8 psB1C3(E,P) | 3 | 9/13 AT(E,P)(DNA purification product) | 1 | (2x)Buffer | 5 | T4 Ligase | 1 | 0 | 10 |
| 9/13 psB1C3(X,P) | 3 | 9/13 AT-His(X,P)(DNA purification product) | 1 | (2x)Buffer | 5 | T4 Ligase | 1 | 0 | 10 |
Miniprep
Honda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/12 SAMmt-YCplac111 | 140 | 1.80 | 1.90 |
| T7-RBS-psB1C3 | 290 | 1.78 | 1.65 |
Liquid Culture
Yasuda
| Sample | Medium |
|---|
| 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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | 9/13 Control (SAMmt) |
| 3 | 9/13 SAMmt (E,P) |
| 4 | - |
| 5 | 9/13 Control (Pcon-psB1C3) |
| 6 | 9/13 Pcon-psB1C3(X,S) |
DNA Purification
Honda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| Pcon-psB1C3(X,S) | 40 | 0.13 | 1.27 |
Electrophoresis
Yasuda
(1)
| Lane | Sample |
|---|
| 1 | 9/12 AT-YCplac111(1) |
| 2 | 9/12 AT-YCplac111(2) |
| 3 | 9/12 AT-YCplac111(3) |
| 4 | 9/12 AT-YCplac111(4) |
| 5 | 9/12 AT-YCplac111(5) |
| 6 | 9/12 AT-YCplac111(6) |
| 7 | 9/12 AT-YCplac111(7) |
| 8 | 9/12 AT-YCplac111(8) |
| 9 | 1kbp GereRuler |
| 10 | 9/12 AT His-YCplac111(1) |
| 11 | 9/12 AT His-YCplac111(2) |
| 12 | 9/12 AT His-YCplac111(3) |
| 13 | 9/12 AT His-YCplac111(4) |
| 14 | 9/12 AT His-YCplac111(5) |
| 15 | 9/12 AT His-YCplac111(6) |
| 16 | 9/12 AT His-YCplac111(7) |
| 17 | 9/12 AT His-YCplac111(8) |
(2)
| Lane | Sample |
|---|
| 1 | 9/12 mamB-YCplac111(1) |
| 2 | 9/12 mamB-YCplac111(2) |
| 3 | 9/12 mamB-YCplac111(3) |
| 4 | 9/12 mamB-YCplac111(4) |
| 5 | 9/12 mamB-YCplac111(5) |
| 6 | 9/12 mamB-YCplac111(6) |
| 7 | 9/12 mamB-YCplac111(7) |
| 8 | 9/12 mamB-YCplac111(8) |
| 9 | 1kbp GereRuler |
| 10 | 9/12 DECARB-YCplac111(1) |
| 11 | 9/12 DECARB-YCplac111(2) |
| 12 | 9/12 DECARB-YCplac111(3) |
| 13 | 9/12 DECARB-YCplac111(4) |
| 14 | 9/12 DECARB-YCplac111(5) |
| 15 | 9/12 DECARB-YCplac111(6) |
| 16 | 9/12 DECARB-YCplac111(7) |
| 17 | 9/12 DECARB-YCplac111(8) |
(3)
| Lane | Sample |
|---|
| 1 | 9/12 DECARB-YCplac111(9) |
| 2 | 9/12 DECARB-YCplac111(10) |
| 3 | 9/12 DECARB-YCplac111(11) |
| 4 | 9/12 DECARB-YCplac111(12) |
| 5 | 9/12 DECARB-YCplac111(13) |
| 6 | 9/12 DECARB-YCplac111(14) |
| 7 | 9/12 DECARB-YCplac111(15) |
| 8 | 9/12 DECARB-YCplac111(16) |
| 9 | 1kbp GereRuler |
| 10 | 9/12 DECARB-YCplac111(17) |
| 11 | 9/12 DECARB-YCplac111(18) |
| 12 | 9/12 DECARB-YCplac111(19) |
| 13 | 9/12 DECARB-YCplac111(20) |
| 14 | 9/12 DiDECARB-YCplac111(1) |
| 15 | 9/12 DiDECARB-YCplac111(2) |
| 16 | 9/12 DiDECARB-YCplac111(3) |
| 17 | 9/12 DiDECARB-YCplac111(4) |
(4)
| Lane | Sample |
|---|
| 1 | 9/12 DiDECARB-YCplac111(5) |
| 2 | 9/12 DiDECARB-YCplac111(6) |
| 3 | 9/12 DiDECARB-YCplac111(7) |
| 4 | 9/12 DiDECARB-YCplac111(8) |
| 5 | 9/12 DiDECARB-YCplac111(9) |
| 6 | 9/12 DiDECARB-YCplac111(10) |
| 7 | 9/12 DiDECARB-YCplac111(11) |
| 8 | 9/12 DiDECARB-YCplac111(12) |
| 9 | 1kbp GereRuler |
| 10 | 9/12 DiDECARB-YCplac111(13) |
| 11 | 9/12 DiDECARB-YCplac111(14) |
| 12 | 9/12 DiDECARB-YCplac111(15) |
| 13 | 9/12 DiDECARB-YCplac111(16) |
| 14 | 9/12 DiDECARB-YCplac111(17) |
| 15 | 9/12 DiDECARB-YCplac111(18) |
| 16 | 9/12 DiDECARB-YCplac111(19) |
| 17 | 9/12 DiDECARB-YCplac111(20) |
Miniprep
Honda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/13 dddD-psB1C3(1) | 645 | 1.82 | 1.70 |
| 9/13 dddD-psB1C3(2) | 660 | 1,69 | 1.74 |
| 9/13 dddD-psB1C3(3) | 635 | 1.87 | 1.57 |
| 9/13 T7-RBS-His-psB1C3(1) | 440 | 1.65 | 1.23 |
| 9/13 T7-RBS-His-psB1C3(2) | 450 | 1.92 | 1.46 |
| 9/13 T7-RBS-His-psB1C3(3) | 190 | 1.44 | 1.26 |
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 | - | 2 | M | 6 | 6 | 38 | 60 |
| 9/14 dddD-psB1C3(2) | 6 | - | 2 | - | 2 | M | 6 | 6 | 38 | 60 |
| 9/14 dddD-psB1C3(3) | 6 | - | 2 | - | 2 | M | 6 | 6 | 38 | 60 |
| 9/14 T7-RBS-His-psB1C3(1) | 5 | - | - | 1 | 1 | H | 3 | - | 18 | 30 |
| 9/14 T7-RBS-His-psB1C3(2) | 5 | - | - | 1 | 1 | H | 3 | - | 18 | 30 |
| 9/14 T7-RBS-His-psB1C3(3) | 10 | - | - | 1 | 1 | H | 3 | - | 15 | 30 |
Dephosphorylation
Honda
| Sample/(µL) | CIAP/(µL) |
|---|
| 9/14 psB1C3(X,S)(gel extraction product) | 20 | 0.25 |
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/14 psB1C3(X,S)(gel extraction product) | 60 | 2.93 | 0.16 |
Ligation
Yasuda
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| Pcon-psB1C3(X,S)(Dephosphorylation product) | 0.83 | 9/12 DECARB(X,S)(DNA purification product) | 2.93 | 10x Lig Buffer | 2 | NEB T4 DNA Ligase | 1 | 13.24 | 20 |
| Pcon-psB1C3(X,S)(Dephosphorylation product) | 0.83 | 9/12 DECARB(X,S)(DNA purification product) | 3.66 | 10x Lig Buffer | 2 | NEB T4 DNA Ligase | 1 | 12.51 | 20 |
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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2~9 | 9/14 AT-psB1C3 |
Gel Extraction
Tatsui
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | 9/14 dddD(µ)-psB1C3(X,P)-(1) |
| 4 | - |
| 5 | 1kb ladder |
| 6 | - |
| 7 | 9/14 dddD(µ)-psB1C3(X,P)-(2) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | - |
| 11 | 9/14 dddD(µ)-psB1C3(X,P)-(3) |
DNA Purification
Tatsui
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/14 dddD(µ)-psB1C3(X,P)-(1) | 1.7 (x50) | 1.37 | 0.33 |
| 9/14 dddD(µ)-psB1C3(X,P)-(2) | 1.6 (x50) | 1.41 | 0.36 |
| 9/14 dddD(µ)-psB1C3(X,P)-(3) | 2.2 (x50) | 1.23 | 0.42 |
| T7-RBS-psB1C3(S cut) | 2.0 (x50) | 2.11 | 0.34 |
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-psB1C3 | 7 | (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-psB1C3 | 25 | (E) | (X) | (S) | (P) | (HorM) | (Buffer) | (BSA) | (MilliQ) | 30 |
| 9/15 T7-RBS-psB1C3 | 25 | (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
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | 9/14 T7-RBS-His-psB1C3(S,P)-(1) |
| 4 | - |
| 5 | 1kb ladder |
| 6 | - |
| 7 | 9/14 T7-RBS-His-psB1C3(S,P)-(1) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | - |
| 11 | 9/14 T7-RBS-His-psB1C3(S,P)-(1) |
DNA Purification
Yasuda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/15 REDOX (PCR product) | 4.9 | 1.76 | 0.66 |
Ligation
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| YCplac111 (SmaI cut)(x3) | 1 | 9/12 AT His (DNA purification product) | 0.3 | (x2)Buffer | 5 | T4 Ligase | 1 | 2.7 | 10 |
| YCplac111 (SmaI cut)(x3) | 1 | 9/15 REDOX (DNA purification product) | 0.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 2.8 | 10 |
| YCplac111 (SmaI cut)(x3) | 1 | 9/12 DECARB(DNA purification product) | 0.4 | (x2)Buffer | 5 | T4 Ligase | 1 | 2.6 | 10 |
| YCplac111 (SmaI cut)(x3) | 1 | 9/12 DiDECARB(DNA purification product) | 0.3 | (x2)Buffer | 5 | T4 Ligase | 1 | 2.7 | 10 |
Liquid Culture
Honda
| Sample | Medium |
|---|
| 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) | 20 | 0.25 |
| 9/13 psB1C3(E,P)(Gel extraction product) | 20 | 0.25 |
| 37°C |
50°C |
75°C |
| 30min |
1h |
15min |
Gel Extraction
Yasuda
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | 9/14 T7-RBS-His-psB1C3(S,P)-(1) |
| 4 | - |
| 5 | - |
| 6 | - |
| 7 | 9/14 T7-RBS-His-psB1C3(S,P)-(2) |
| 8 | - |
| 9 | - |
| 10 | - |
| 11 | 9/14 T7-RBS-His-psB1C3(S,P)-(3) |
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/14 T7-RBS-His-psB1C3(S,P)-(1) | 25 | 1.81 | 0.50 |
| 9/14 T7-RBS-His-psB1C3(S,P)-(2) | 25 | 1.13 | 0.21 |
| 9/14 T7-RBS-His-psB1C3(S,P)-(3) | 25 | 1.65 | 0.09 |
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 | - | - | 3 | 3 | H | 9 | - | 61.37 | 90 |
| Control | 0.3 | - | - | - | - | H | 1 | - | 8.9 | 10 |
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) | 2.0 | 9/15 dddD(µ)(X,P)(Gel extraction product) | 2.0 | (x2)Buffer | 5 | T4 Ligase | 1 | 0 | 10 |
| 9/15 psB1C3 (E,P)(Dephosphorylation product) | 0.4 | 9/12 SAMmt (E,P)(DNA purification product) | 2.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.7 | 10 |
| 9/15 psB1C3 (E,P)(Dephosphorylation product) | 1.3 | 9/15 REDOX (E,P)(DNA purification product) | 2.4 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.3 | 10 |
| 9/15 T7-RBS(S,P)(Gel extraction product) | 2.6 | 9/15 AT His (X,P)(DNA purification product) | 1.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.2 | 10 |
Transformation
Murata and Yasuda
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| DECARB-YCplac111 | 2 | DH5&alfa; | 10 | 12 | SOD |
| DiDECARB-YCplac111 | 2 | DH5&alfa; | 10 | 12 | SOD |
| dddD(µ)-T7-RBS-psB1C3 | 2 | DH5&alfa; | 10 | 12 | SOD |
| SAMmt-psB1C3 | 2 | DH5&alfa; | 10 | 12 | SOD |
| REDOX-psB1C3 | 2 | DH5&alfa; | 10 | 12 | SOD |
| AT-T7-RBS-psB1C3 | 2 | DH5&alfa; | 10 | 12 | SOD |
DNA Purification
Yasuda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/15 REDOX (E,P) | 45 | - | - |
9/16
Miniprep
Yasuda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/15 AT-psB1C3-(1) | 280 | 1.81 | 2.08 |
| 9/15 AT-psB1C3-(1) | 205 | 1.64 | 1.32 |
| 9/15 AT-psB1C3-(1) | 320 | 1.66 | 1.61 |
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)
| Lane | Sample |
|---|
| 1-8 | 9/15 REDOX-YCplac111 |
| 9 | 1kb ladder |
| 10-17 | 9/15 DECARB-YCplac111 |
(2)
| Lane | Sample |
|---|
| 1-8 | 9/15 DiDECARB-YCplac111 |
| 9 | 1kb ladder |
| 10-17 | 9/15 AT-His-YCplac111 |
(3)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2-8 | 9/15 AT-His-YCplac111 |
(4)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2-6 | 9/15 AT-His-YCplac111 |
Gel Extraction
Yamauchi
(1)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | (written in "magnetosome") |
| 4 | - |
| 5 | 1kb ladder |
| 6 | - |
| 7 | (written in "magnetosome") |
| 8 | - |
| 9 | 1kb ladder |
| 10 | - |
| 11 | 9/15 psB1C3(E,P) |
(2)
| Lane | Sample |
|---|
| 1 | 1kb ladder |
| 2 | - |
| 3 | 9/15 T7-RBS-psB1C3(P) |
| 4 | - |
| 5 | 1kb ladder |
| 6 | - |
| 7 | 9/15 T7-RBS-psB1C3(S,P) |
| 8 | - |
| 9 | 1kb ladder |
| 10 | - |
| 11 | 9/15 T7-RBS-psB1C3(S,P) |
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 7. 9/15 T7-RBS-psB1C3(S,P) | 90 | 1.40 | 0.44 |
(3)
| Lane | Sample |
|---|
| 1 | - |
| 2 | 1kb ladder |
| 3 | - |
| 4 | 9/15 T7-RBS-psB1C3(S,P) |
| 5 | - |
| 6 | 1kb ladder |
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 4. 9/15 T7-RBS-psB1C3(S,P) | 80 | 1.30 | 1.02 |
DNA Purification
Yasuda
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/15 T7-RBS-psB1C3(S,P)(Dephosphorylation product) | 0.7(x50) | 1.81 | 1.13 |
| 9/15 psB1C3(E,P)(Dephosphorylation product) | 1.1(x50) | 1.59 | 1.25 |
Ligation
Yasuda
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| 9/16 T7-RBS(S,P)(DNA purification product) | 1.7 | 9/15 dddD(µ)(X,P)(gel extraction product) | 2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.3 | 10 |
| 9/16 psB1C3 (E,P)(DNA purification product) | 0.55 | 9/15 SAMmt (E,P)(gel extraction product) | 2.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.55 | 10 |
| 9/16 psB1C3 (E,P)(DNA purification product) | 1.6 | 9/15 REDOX (E,P)(gel extraction product) | 2.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.2 | 10 |
| 9/16 T7-RBS-His(S,P)(DNA purification product) | 2.6 | 9/15 AT His(X,P)(gel extraction product) | 1.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.2 | 10 |
Dephosphorylation
Murata
| Sample/(µL) | CIAP | Total |
|---|
| 9/13 psB1C3 (X,S)(gel extraction product) | 16 | 0.2 | 16.2 |
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
| Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
|---|
| 9/16 psB1C3(X,S)(Dephosphorylation product) | 35 | 1.69 | 0.56 |
Liquid Culture
Yasuda
| Sample | Medium |
|---|
| 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) | 8.2 | 0.5 | - | - | 0.5 | H | 1.5 | 0 | 4.3 | 15 |
| 9/15 AT-psB1C3-(3)(Miniprep product) | 9.8 | - | 1 | - | 1 | M | 3 | 3 | 12.2 | 30 |
| Control | 0.5 | - | - | - | - | M | 1 | 1 | 7.5 | 10 |
Ligation
Honda
| Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) |
|---|
| psB1C3 | 1.1 | 9/12 DECARB(X,E)(DNA purification product) | 2.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0 | 10 |
| psB1C3 | 0.85 | 9/12 DiDECARB(X,E)(DNA purification product) | 2.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.95 | 10 |
Transformation
Honda
| Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium |
|---|
| 9/16 DECARB-psB1C3 | 1 | DH5α | 10 | 11 | - |
| 9/16 DiDECARB-psB1C3 | 1 | DH5α | 10 | 11 | - |
15min on ice - 1min heat shock(42°C) - 45min preculture (42°C)
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