Template:Kyoto/Notebook/DMS

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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 コンピ名 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 Tag/(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

@

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 @ 1.37 0.42
dddD PCR product 35 1.54 0.85

8/4

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

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

(2)

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

(3)

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

(4)

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

control(2)

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

(4)

Quick Tag/(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 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 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 @
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 @ 50 61.2 SOC
8/19 Ligation (2) 11.2 @ 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 @ 20 25 SOC
control 5 @ 20 25 SOC

@

8/21

Colony PCR

Honda,Nakamura

Master Mix

Quick Tag/(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
@ @

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 @ 50 60 SOC
8/25 Ligation(2) 10 @ 50 60 SOC
8/25 Ligation(3) 10 @ 30 40 SOC

@

8/27

Colony PCR

Murata

Master Mix

Quick Tag/(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

Quick Tag/(µL) VF2/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
@ @ @ @ 25
Predenature Denature Annealing Extension
95°C 98°C 65°C 72°C
5min 20sec 15sec 50sec

Denature-Annealing-Extension @cycle

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 @ SOC

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