From 2013.igem.org
15-08-2013
Amplification from SR_01 to FS_23; 6.2 kb
Amplification of DelG; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelG; run at 100 V, 0.8 % gel (TAE)(14.08)
- Reaction (5x20 µl)
| what | µl
|
| D. acidovorans SPH-1 colony | 1
|
| SR_01: (1/10) | 2
|
| FS_23: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 4
|
- Conditions
| Biorad T100
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 66 ↓ 0.5 | 5
|
| 72 | 3:20
|
| 18 | 98 | 1
|
| 64 | 5
|
| 72 | 3:20
|
| 1 | 72 | 10min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelG was successful
- Bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- Amplification will be repeated to increase the concentration for the Gibson Assembly
16-08-2013
Concentration measurement
the fragment was gel purified
| Fragment | Primer | Date PCR | Concentration
|
| DelG | SR01-FS23 | 15-08-2013 | 52.6 ng/µl
|
| DelG after nucleotide removal | SR01-FS23 | 15-08-2013 | 30 ng/µl
|
Amplification from SR_01 to FS_23; 6.2 kb
Amplification of DelG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelG ; run at 100 V, 0.8 % gel (TAE)(14.08)
- Reaction (5x20 µl)
| what | µl
|
| D. acidovorans SPH-1 colony | 1
|
| SR_01: (1/10) | 2
|
| FS_23: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 4
|
- Conditions
| Biorad T100
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 66 ↓ 0.5 | 5
|
| 72 | 3:20
|
| 18 | 98 | 1
|
| 64 | 5
|
| 72 | 3:20
|
| 1 | 72 | 10min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelG was successful
- Bands were cut out and DNA purified using QIAquick Gel Extraction Kit
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