From 2014.igem.org
(Difference between revisions)
|
|
| (One intermediate revision not shown) |
| Line 1,392: |
Line 1,392: |
| | </tr> | | </tr> |
| | </table> | | </table> |
| | + | |
| | + | <h4>DNA Extraction</h4> |
| | + | <span class="kyoto-author">Yasuda and Murata</span> |
| | + | |
| | + | <p>Nuelei Lysis Solution Solution 600µL pipeting 85°C 5min incuvate and cooling</p> |
| | + | <p>RNase Solution 3µL invert(2-5times) 37°C 45min incuvate and cooling</p> |
| | + | <p>Protein Preciprtation Solution 200µL Highspeed voltex on ice 5min and 16000g 3min (Supernatant ladled)</p> |
| | + | <p>isopropyl alcohol 600µL invert 16000g 2min (Supernatant discarded)</p> |
| | + | <p>70%ethanol 600µL invert 16000g 2min aspirate 10-15min air-drying</p> |
| | + | <p>DNA Rehydration Solution 100µL 65°C 1h incubate</p> |
| | | | |
| | <h4>Restriction Enzyme Digestion</h4> | | <h4>Restriction Enzyme Digestion</h4> |
| Line 2,048: |
Line 2,058: |
| | </tr> | | </tr> |
| | </table> | | </table> |
| | + | <p>Vector and insert are elaborated.</p> |
| | | | |
| | <h4>Transformation</h4> | | <h4>Transformation</h4> |
| Line 2,332: |
Line 2,343: |
| | </tr> | | </tr> |
| | </table> | | </table> |
| | + | <img src="https://static.igem.org/mediawiki/2014/thumb/7/7c/Kyoto-Img022.jpg/586px-Kyoto-Img022.jpg"> |
| | | | |
| | <h4>Transformation</h4> | | <h4>Transformation</h4> |
| Line 2,511: |
Line 2,523: |
| | </tr> | | </tr> |
| | </table> | | </table> |
| | + | <img src="https://static.igem.org/mediawiki/2014/thumb/9/9f/Kyoto-Img023.jpg/648px-Kyoto-Img023.jpg"> |
| | | | |
| | </div> | | </div> |
Latest revision as of 04:55, 13 October 2014
2/10
Restriction Enzyme Digestion
Yasuda
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| J23100 |
5.7 |
- |
- |
0.5 |
0.5 |
10xBuffer(K) |
3 |
- |
20.3 |
30 |
| RBS-GFP-DT |
7.5 |
- |
0.5 |
- |
0.5 |
10xBuffer(M) |
3 |
3 |
15.5 |
30 |
Electrophoresis/Gel Extraction
Yasuda
| Lane |
Sample |
| 1 |
1kbp ladder(2µL) |
| 2 |
J23100 DNA control |
| 3 |
J23100 S,P experimental |
| 4 |
J23100 S,P experimental |
| Lane |
Sample |
| 1 |
1kbp ladder(2µL) |
| 2 |
RBS-GFP-DT DNA control |
| 3 |
RBS-GFP-DT X,P experimental |
| 4 |
RBS-GFP-DT X,P experimental |
Restriction Enzyme Digestion
Yasuda
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| J23100 |
10 |
- |
- |
0.5 |
0.5 |
10xBuffer(K) |
3 |
- |
16 |
30 |
2/12
Electrophoresis/Gel Extraction
Yasuda
| Lane |
Sample |
| 1 |
1kbp ladder(2µL) |
| 2 |
J23100 DNA control |
| 3 |
J23100 S,P experimental |
| 4 |
J23100 S,P experimental |
| Sample |
Concentration/(µg/mL) |
260/280 |
260/230 |
| GFP |
16.7 |
1.46 |
0.64 |
| Pcon |
17.2 |
1.69 |
0.26 |
Ligation
Inoue
| Vector/(µL) |
Insert/(µL) |
Buffer/(µL) |
Ligase/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 2/10 J23100 S,P 2100bp |
5.8 |
2/10 RBS-GFP-DT 898bp |
4.9 |
2x Buffer |
5 |
T4 Ligase |
3 |
- |
10 |
Vector and insert are evaporated
Transformation
Yasuda
| Sample/(µL) |
CompetentCells/(µL) |
Total/(µL) |
Medium |
| 2/12 J23119(A) Pcon |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
SOC |
| 2/12 I712074 (Ak) T7 |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
SOC |
| 2/12 Ligation Pcon+RBS-GFP-PT |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
SOC |
2/13
Colony PCR
Yasuda,Murakami
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 30 |
1.2 |
1.2 |
27.6 |
60 |
| Predenature |
Denature |
Annealing |
Extension |
| 94°C |
94°C |
55°C |
68°C |
| 5min |
30sec |
30sec |
72sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
Pcon-RBS-GFP-DT (1) |
| 2 |
Pcon-RBS-GFP-DT (2) |
| 3 |
Pcon-RBS-GFP-DT (3) |
| 4 |
Pcon-RBS-GFP-DT (4) |
| 5 |
T7 |
Electrophoresis
Honda
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
2/12 Pcon-RBS-GFP-DT (1) |
| 3 |
2/12 Pcon-RBS-GFP-DT (2) |
| 4 |
2/12 Pcon-RBS-GFP-DT (3) |
| 5 |
2/12 Pcon-RBS-GFP-DT (4) |
| 6 |
2/12 I712074(Ak) T7 |
Liquid Culture
No Name
| Sample |
Medium |
| Pcon-RBS-GFP-DT |
Amp |
2/14
Miniprep
Murakami
| Sample |
| 2/13 Pcon-RBS-GFP-DT(1) |
| 2/13 Pcon-RBS-GFP-DT(2) |
2/23
Self-Making Competent Cells (DH5α)
Tatsui and Kojima
3/4
Restriction Enzyme Digestion
Kato, Murata and Shimazaki
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 9/8 J23100 325µg/ml |
6 |
- |
- |
0.5 |
0.5 |
H |
3 |
3 |
17 |
30 |
| 9/8 J23100 325µg/ml control |
0.3 |
- |
- |
- |
- |
H |
1 |
1 |
7.7 |
10 |
| 9/28 RBS-GFP-DT 215.9µg/ml |
9.3 |
- |
0.5 |
- |
0.5 |
H |
3 |
3 |
13.7 |
30 |
| control |
0.5 |
- |
- |
- |
- |
H |
1 |
1 |
7.5 |
10 |
Electrophoresis
Kato, Murata and Shimazaki
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
9/8 J23100 S,P |
| 3 |
9/8 J23100 control |
| 4 |
9/28 RBS-GFP DT X,P |
| 5 |
9/28 RBS-GFP DT control |
Gel Extraction
Kato, Murata and Shimazaki
| Lane |
Sample |
| 1 |
9/8 J23100 S,P |
| 2 |
Blank |
| 3 |
Blank |
| 4 |
9/28 RBS-GFP-DT X,P |
| 5 |
1kbp ladder |
3/5
Ligation
Kato, Shimazaki and Murata
| Vector/(µL) |
Insert/(µL) |
Buffer/(µL) |
Ligase/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 3/4 J23100 S,P |
4.8 |
3/4 RBS-GFP-DT X,P |
9.2 |
Reaction Buffer |
2 |
Ligase(Bio Lub) |
1 |
3 |
20 |
Transformation
No Name
| Sample/(µL) |
CompetentCells/(µL) |
Total/(µL) |
Medium |
| 3/5 J23119 |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
SOC |
| 3/5 I712074 |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
SOC |
| 3/5 ligation Pcon RBS-GFP-DT |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
SOC |
3/6
Colony PCR
Kato and Shimazaki
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 65 |
2.6 |
2.6 |
59.8 |
130 |
| Predenature |
Denature |
Annealing |
Extension |
| 94°C |
94°C |
55°C |
68°C |
| 5min |
30sec |
30sec |
72sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
3/5 J23119 (1) |
| 2 |
3/5 J23119 (2) |
| 3 |
3/5 J23119 (3) |
| 4 |
3/5 J23119 (4) |
| 5 |
3/5 I712074 (1) |
| 6 |
3/5 I712074 (2) |
| 7 |
3/5 I712074 (3) |
| 8 |
3/5 I712074 (4) |
| Number |
Sample |
| 1 |
3/5 Pcon RBS-GFP-DT (1) |
| 2 |
3/5 Pcon RBS-GFP-DT (2) |
| 3 |
3/5 Pcon RBS-GFP-DT (3) |
| 4 |
3/5 Pcon RBS-GFP-DT (4) |
Electrophoresis
Murata
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
3/5 J23119 (1) |
| 3 |
3/5 J23119 (2) |
| 4 |
3/5 J23119 (3) |
| 5 |
3/5 J23119 (4) |
| 6 |
3/5 I712074 (1) |
| 7 |
3/5 I712074 (2) |
| 8 |
3/5 I712074 (3) |
| 9 |
3/5 I712074 (4) |
| 10 |
3/5 Pcon RBS-GFP-DT (1) |
| 11 |
3/5 Pcon RBS-GFP-DT (2) |
| 12 |
3/5 Pcon RBS-GFP-DT (3) |
| 13 |
3/5 Pcon RBS-GFP-DT (4) |
| 14 |
1kbp ladder |
3/7
Miniprep
Murata
| Sample |
| 3/6 Pcon RBS-GFP-DT (1) |
| 3/6 Pcon RBS-GFP-DT (2) |
3/10
Transformation
Okazaki
| Sample/(µL) |
CompetentCells/(µL) |
Medium |
| 3/10 RBS-GFP |
Self-Making Competent Cells (DH5α) |
CP |
| RBS |
Self-Making Competent Cells (DH5α) |
Amp |
| DT |
Self-Making Competent Cells (DH5α) |
CP |
| 3/10 T7 |
Self-Making Competent Cells (DH5α) |
Amp |
3/11
Colony PCR
Shimazaki
Master Mix
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 50 |
2 |
2 |
46 |
100 |
| Predenature |
Denature |
Annealing |
Extension |
| 97°C |
94°C |
55°C |
68°C |
| 5min |
30sec |
30sec |
60sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
3/10 RBS (1) |
| 2 |
3/10 RBS (2) |
| 3 |
3/10 pSB1C3 (1) |
| 4 |
3/10 pSB1C3 (2) |
| 5 |
3/10 DT (1) |
| 6 |
3/10 DT (2) |
| 7 |
3/10 T7 (1) |
| 8 |
3/10 T7 (2) |
| 9 |
control |
3/12
Electrophoresis
Shimazaki
| Lane |
Sample |
| 1 |
3/10 RBS (1) |
| 2 |
3/10 RBS (2) |
| 3 |
3/10 pSB1C3 (1) |
| 4 |
3/10 pSB1C3 (2) |
| 5 |
3/10 DT (1) |
| 6 |
3/10 DT (2) |
| 7 |
3/10 T7 (1) |
| 8 |
3/10 T7 (2) |
| 9 |
control |
| 10 |
1kbp ladder |
Colony PCR
Shimazaki
Master Mix
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 50 |
2.0 |
2.0 |
46 |
100 |
| Predenature |
Denature |
Annealing |
Extension |
| 97°C |
94°C |
55°C |
68°C |
| 5min |
30sec |
30sec |
60sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
3/10 RBS 3 |
| 2 |
3/10 RBS 4 |
| 3 |
3/10 pSB1C3 4 |
| 4 |
3/10 pSB1C3 5 |
| 5 |
3/10 DT 4 |
| 6 |
3/10 DT 5 |
| 7 |
3/10 T7 3 |
| 8 |
3/10 T7 4 |
| 9 |
new QuickTaq control |
| 10 |
old QuickTaq control |
3/14
Electrophoresis
Kojima
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
3/10 RBS (1) |
| 3 |
3/10 RBS (2) |
| 4 |
3/10 pSB1C3 (3) |
| 5 |
3/10 pSB1C3 (4) |
| 6 |
3/10 DT (5) |
| 7 |
3/10 DT (6) |
| 8 |
3/10 T7 (7) |
| 9 |
3/10 T7 (8) |
| 10 |
old Quick Taq control (9) |
| 11 |
new Quick Taq control (10) |
| 12 |
1kbp ladder |
Colony PCR
Kojima
Master Mix
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 17.5 |
0.7 |
0.7 |
16.1 |
35 |
| Predenature |
Denature |
Annealing |
Extension |
| 97°C |
95°C |
58°C |
68°C |
| 5min |
30sec |
30sec |
30sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
RBS-4 |
| 2 |
DT-5 |
| 3 |
control |
Electrophoresis
Kojima
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
RBS-4 |
| 3 |
DT-5 |
| 4 |
control |
Colony PCR
Yasuda and Shimazaki
Master Mix
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 17.5 |
0.7 |
0.7 |
16.1 |
35 |
| Predenature |
Denature |
Annealing |
Extension |
| 97°C |
95°C |
58°C |
68°C |
| 5min |
30sec |
30sec |
60sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
pSB1C3-3 |
| 2 |
T7-5 |
| 3 |
control |
Electrophoresis
Shimazaki
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
pSB1C3-3 |
| 3 |
T7-5 |
| 4 |
control |
| 4 |
2/10 MilliQ |
3/15
Liquid Culture
Yasuda and Murata
| Sample |
Medium |
| Ruegeria pomeroyi fungus
liquid |
YTSS medium |
3/17
Liquid Culture
Yoshida
| Sample |
Medium |
| 3/10 pSB1C3-3 |
CP |
| 3/10 DT-5 |
CP |
| 3/10 T7-5 |
Amp |
| 3/10 RBS-4 |
Amp |
3/18
Miniprep
Murata and Yasuda
| Sample |
Concentration/(µg/mL) |
260/280 |
260/230 |
| 3/17 DT CP |
64.4 |
1.17 |
0.29 |
| 3/17 RBS Amp |
61.4 |
1.28 |
0.36 |
| 3/17 T7 Amp |
66.8 |
1.22 |
0.34 |
| 3/17 pSB1C3 |
64.2 |
1.31 |
0.39 |
DNA Extraction
Yasuda and Murata
Nuelei Lysis Solution Solution 600µL pipeting 85°C 5min incuvate and cooling
RNase Solution 3µL invert(2-5times) 37°C 45min incuvate and cooling
Protein Preciprtation Solution 200µL Highspeed voltex on ice 5min and 16000g 3min (Supernatant ladled)
isopropyl alcohol 600µL invert 16000g 2min (Supernatant discarded)
70%ethanol 600µL invert 16000g 2min aspirate 10-15min air-drying
DNA Rehydration Solution 100µL 65°C 1h incubate
Restriction Enzyme Digestion
Yoshida
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3 E,S 3/18 miniprep product |
20 |
1 |
1 |
- |
- |
M |
3 |
3 |
2 |
30 |
| control |
3 |
- |
- |
- |
- |
M |
1 |
1 |
5 |
10 |
Cloning PCR
Murata and Yasuda
| 2K KAPA HiFi HotStart ReadyMix/(µL) |
F dddD 66/(µL) |
R dddD 65/(µL) |
R.pomeroyi Genome/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 12.5 |
0.75 |
0.75 |
3 |
8 |
25 |
| Predenature |
Denature |
Annealing |
Extension |
Final Extension |
| 95°C |
98°C |
65°C |
72°C |
72°C |
| 3min |
20sec |
15sec |
150sec |
1sec |
Denature-Annealing-Extension 25cycle
3/19
Electrophoresis
Yoshida
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
pSB1C3 E,X |
| 3 |
pSB1C3 control |
Restriction Enzyme Digestion
Yoshida
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3 3/18 miniprep product |
20 |
1 |
- |
1 |
- |
H |
3 |
- |
5 |
30 |
| control |
3 |
- |
- |
- |
- |
H |
1 |
- |
6 |
10 |
Electrophoresis
Kojima
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
dddD PCR product |
PCR
Kojima
| 3/19 DNA Purification product/(µL) |
Fw/(µL) |
Rv/(µL) |
HiFi Mix/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 1 |
0.75 |
0.75 |
12.5 |
10 |
25 |
| R.pomeroyi Genome/(µL) |
Fw/(µL) |
Rv/(µL) |
HiFi Mix/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 3 |
0.75 |
0.75 |
12.5 |
8 |
25 |
| Predenature |
Denature |
Annealing |
Extension |
Final Extension |
| 95°C |
98°C |
68°C |
72°C |
72°C |
| 5min |
20sec |
15sec |
90sec |
5min |
Denature-Annealing-Extension 25cycle
3/20
Electrophoresis
Kojima
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
pSB1C3 E,S |
| 3 |
control |
| 4 |
dddD(1) 3/19 PCR product |
| 5 |
dddD(2) 3/19 PCR product |
Gel Extraction
Yasuda and Tatsui
| Lane |
Sample |
| 1 |
Blank |
| 2 |
1kbp ladder |
| 3 |
pSB1C3 |
| 4 |
Blank |
| 5 |
pSB1C3 |
Gel Extraction
Murara
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
dddD |
| 3 |
dddD |
| Sample |
Concentration/(µg/mL) |
260/280 |
260/230 |
| dddD |
35 |
1.63 |
0.85 |
3/24
Restriction Enzyme Digestion
Honda
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3 |
11 |
0.5 |
0.5 |
- |
- |
M |
1.5 |
1.5 |
- |
15 |
| dddD |
12.5 |
0.5 |
- |
0.5 |
- |
M |
1.5 |
- |
- |
15 |
PCR
Honda
| 2x KAPA HiFi HotStart ReadyMix/(µL) |
F dddD 62/(µL) |
R dddD 68/(µL) |
Templete 3/24 dddD/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 12.5 |
0.75 |
0.75 |
pick up |
11 |
25 |
| Predenature |
Denature |
Annealing |
Extension |
Final Extension |
| 95°C |
98°C |
68°C |
72°C |
72°C |
| 5min |
20sec |
15sec |
90sec |
5min |
Denature-Annealing-Extension 25cycle
| Sample |
Concentration/(µg/mL) |
260/280 |
260/230 |
| dddD |
62.7 |
1.77 |
1.88 |
Ligation
Honda
| Vector/(µL) |
Insert/(µL) |
Buffer/(µL) |
Ligase/(µL) |
MilliQ/(µL) |
Total/(µL) |
| dddD |
1.3 |
pSB1C3 |
0.43 |
2xBuffer |
5 |
T4Ligase |
1 |
2.27 |
10 |
Liquid Culture
Tatsui
| Sample |
Medium |
| pSB1C3(3) |
CP |
| pSB1C3(4) |
CP |
3/25
Miniprep
Tatsui
| Sample |
Concentration/(µg/mL) |
| pSB1C3 3/24 (3) |
41 |
| pSB1C3 3/24 (4) |
57 |
Restriction Enzyme Digestion
Murata
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3 3/24 (3) |
15.4 |
0.5 |
- |
0.5 |
- |
H |
3 |
- |
10.6 |
30 |
| 3/24 PCR product dddD E,S |
3.2 |
0.5 |
- |
0.5 |
- |
H |
3 |
- |
22.8 |
30 |
Gel Extraction
Murata
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
pSB1C3(3) E,S 3/25 |
| 3 |
Blank |
| 4 |
Blank |
| 5 |
Blank |
| 6 |
dddD 3/25 E,S |
| 7 |
dddD 3/25 E,S |
| Sample |
Concentration/(µg/mL) |
260/280 |
260/230 |
| dddD |
4.9 |
17.5 |
0.52 |
| pSB1C3 |
2.0 |
- |
0.21 |
3/26
Ligation
Yasuda
| Vector/(µL) |
Insert/(µL) |
Buffer/(µL) |
Ligase/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3 2047bp |
30 |
dddD 2545bp |
30 |
2x Rapid Ligation Buffer |
5 |
T4Ligase |
3 |
- |
10 |
Vector and insert are elaborated.
Transformation
Yasuda
3/27
Restriction Enzyme Digestion
Murata
| Sample/(µL) |
EcoR1/(µL) |
Xba1/(µL) |
Spe1/(µL) |
Pst1/(µL) |
Buffer/(µL) |
BSA/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3(4) 3/25 |
20.1 |
0.5 |
- |
0.5 |
- |
10xBuffer |
3 |
- |
5.9 |
30 |
| dddD 3/24 PCR product |
3.2 |
0.5 |
- |
0.5 |
- |
10xBuffer |
3 |
- |
22.8 |
30 |
Gel Extraction
Honda
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
pSB1C3 E,S 3/27 |
| 3 |
pSB1C3 E,S 3/27 |
| 4 |
Blank |
| 5 |
dddD E,S 3/27 |
| 6 |
dddD E,S 3/27 |
| Sample |
Concentration/(µg/mL) |
260/280 |
260/230 |
| pSB1C3 |
9 |
1.58 |
0.04 |
| dddD |
16.0 |
1.78 |
0.08 |
Ligation
Honda
| Vector/(µL) |
Insert/(µL) |
Buffer/(µL) |
Ligase/(µL) |
MilliQ/(µL) |
Total/(µL) |
| pSB1C3 |
11 |
dddD |
15 |
2x Rapid Ligation Buffer |
5 |
T4 DNA Ligase |
1 |
4 |
10 |
3/28
PCR
Yasuda
| 2x KAPA HiFi HotStart ReadyMix/(µL) |
F Sequence primer/(µL) |
R Spe1-dddD/(µL) |
Templete/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 12.5 |
0.75 |
0.75 |
pick up |
11 |
25 |
| Predenature |
Denature |
Annealing |
Extension |
Final Extension |
Cooling |
| 95°C |
98°C |
68°C |
72°C |
72°C |
| 5min |
20sec |
15sec |
90sec |
5min |
Denature-Annealing-Extension 25cycle
| Number |
Sample |
| 1 |
Fw:Sequence primer (1) 57.9/Rv:Spe1 dddD 2276bp |
| 2 |
Fw:Sequence primer (2) 57.3/Rv:Spe1 dddD 1907bp |
| 3 |
Fw:Sequence primer (3) 57.6/Rv:Spe1 dddD 1527bp |
| 4 |
Fw:Sequence primer (4) 56.3/Rv:Spe1 dddD 1189bp |
| 5 |
Fw:Sequence primer (5) 57.2/Rv:Spe1 dddD 799bp |
| 6 |
Fw:Sequence primer (6) 56.4/Rv:Spe1 dddD 398bp |
| 7 |
Fw:Sequence primer VF2 56.4/Rv:Spe1 dddD 266bp |
| 8 |
Fw:pre-dddD PCR product 62/Rv:Spe1 dddD |
| 9 |
Fw:Blank/Rv:Spe1 dddD |
Electrophoresis
Murata
| Lane |
Sample |
| 1 |
1kbp ladder |
| 2 |
Seaquence primer (1) PCR product |
| 3 |
Seaquence primer (2) PCR product |
| 4 |
Seaquence primer (3) PCR product |
| 5 |
Seaquence primer (4) PCR product |
| 6 |
Seaquence primer (5) PCR product |
| 7 |
Seaquence primer (6) PCR product |
| 8 |
Seaquence primer VF2 |
| 9 |
F pre-dddD cloning |
| 10 |
Blank |
| 11 |
1kbp ladder |
Transformation
Kojima
| Sample/(µL) |
CompetentCells/(µL) |
Total/(µL) |
Medium |
| dddD |
1 |
Self-Making Competent Cells (DH5α) |
10 |
11 |
CP |
3/30
Colony PCR
Yasuda
| Quick Taq/(µL) |
VF/(µL) |
VR/(µL) |
MilliQ/(µL) |
Total/(µL) |
| 5 |
0.2 |
0.2 |
4.6 |
10 |
| Predenature |
Denature |
Annealing |
Extension |
| 97°C |
95°C |
58°C |
68°C |
| 5min |
30sec |
30sec |
168sec |
Denature-Annealing-Extension 30cycle
| Number |
Sample |
| 1 |
3/26 dddD(pSB1C3) (1) |
| 2 |
3/26 dddD(pSB1C3) (2) |
| 3 |
3/26 dddD(pSB1C3) (3) |
| 4 |
3/26 dddD(pSB1C3) (4) |
| 5 |
3/26 dddD(pSB1C3) (5) |
| 6 |
3/28 dddD(pSB1C3) (6) |
| 7 |
3/28 dddD(pSB1C3) (7) |
| 8 |
3/28 dddD(pSB1C3) (8) |
| 9 |
3/28 dddD(pSB1C3) (9) |
| 10 |
3/28 dddD(pSB1C3) (10) |
3/31
Electrophoresis
Yasuda
| Number |
Sample |
| 1 |
1kbp ladder |
| 2 |
3/26 dddD(pSB1C3) (1) |
| 3 |
3/26 dddD(pSB1C3) (2) |
| 4 |
3/26 dddD(pSB1C3) (3) |
| 5 |
3/26 dddD(pSB1C3) (4) |
| 6 |
3/26 dddD(pSB1C3) (5) |
| 7 |
1kbp ladder |
| 8 |
3/28 dddD(pSB1C3) (6) |
| 9 |
3/28 dddD(pSB1C3) (7) |
| 10 |
3/28 dddD(pSB1C3) (8) |
| 11 |
3/28 dddD(pSB1C3) (9) |
| 12 |
3/28 dddD(pSB1C3) (10) |
| 13 |
1kbp ladder |
"
