Template:Kyoto/Notebook/DMS

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Revision as of 14:44, 2 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 placIII (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)
YCplacIII1dddD1(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)

Retrieved from "http://2014.igem.org/Template:Kyoto/Notebook/DMS"