Template:Kyoto/Notebook/DMS
From 2014.igem.org
(Difference between revisions)
Line 2,140: | Line 2,140: | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
<tr><td>1</td><td>1kb ladder</td> | <tr><td>1</td><td>1kb ladder</td> | ||
- | <tr><td>2</td><td>8/29 | + | <tr><td>2</td><td>8/29 dddD(DNA purification product)1µL+MilliQ 5µL+looding dye(x6)1µL</td> |
<tr><td>3</td><td>control</td> | <tr><td>3</td><td>control</td> | ||
</table> | </table> |
Revision as of 06:37, 30 September 2014
7/31
Restriction Enzyme Digestion
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
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
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
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
pSB1C3-dddD | 3 | コンピ名 | 30 | 33 | SOC |
8/2
PCR Cloning
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
8/2 Pre-dddD-Suf PCR product
Colony PCR
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
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
dddD PCR product | 245 | 1.59 | 2.17 |
Restriction Enzyme Digestion
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
@
Lane | Sample |
---|---|
1 | pSB1C3-dddD(1) |
2 | pSB1C3-dddD(2) |
3 | pSB1C3-dddD(3) |
4 | 1kb ladder (3µL) |
5 | 1kb ladder (1µL) |
Gel Extraction
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
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
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
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
Lane | Sample |
---|---|
1 | 1kbp ladder |
2 | (1) |
3 | (2) |
4 | (3) |
5 | (4) |
8/18
PCR
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
Number | Sample |
---|---|
1 | 1kbp ladder |
2 | (1) |
3 | (2) |
4 | (3) |
5 | (4) |
Restriction Enzyme Digestion
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
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
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
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
(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
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
(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
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
dddD+pSB1C3 | 5 | @ | 20 | 25 | SOC |
control | 5 | @ | 20 | 25 | SOC |
@
8/21
Colony PCR
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