From 2013.igem.org
(Difference between revisions)
Revision as of 10:28, 1 October 2013
03-07-2013
Amplification from FS_06 to FS_09; 8.5 kb
Amplification of DelA-F and DelF-G run at 130 V, 0.8 % gel (TAE)
Gel after removing the DelA-F-fragment.
- Reaction
| µl 1st PCR | what | µl 2nd PCR
|
| 1 | D. acidovorans DSM-39 | 1
|
| 2.5 | FS_06 (1/10) | 2.5
|
| 2.5 | FS_09 (1/10) | 2.5
|
| 25 | Phusion Master Mix | 25
|
| - | DMSO | 2.5
|
| 19 | dd H2O | 19
|
- Conditions
| Biorad MyCycler
|
| Cycles-PCR | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 72 | 2:30 min
|
| 1 | 72 | 10 min
|
| 1 | 4 | inf
|
Results:
- Amplification of DelFG did not work
- PCR will be repeated with a lower annealing temperature of 68°C (touchdown)
Amplification from FS_06 to FS_09; 8.5 kb
Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane)
Gel after removing DelA-F-fragment
- Reaction
| what | µl 2nd PCR
|
| D. acidovorans DSM-39 | 1
|
| FS_06 (1/10) | 2.5
|
| FS_09 (1/10) | 2.5
|
| Phusion Master Mix | 25
|
| DMSO | 2.5
|
| dd H2O | 16.5
|
- Conditions
| Biorad MyCycler
|
| Cycles-PCR | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 68 ↓ 0.5 | 5
|
| 72 | 3 min
|
| 18 | 98 | 1
|
| 66 | 5
|
| 72 | 3 min
|
| 1 | 72 | 10 min
|
| 1 | 4 | inf
|
Results:
- Amplification of DelFG did not work, many different side product occured, PCR will be repeated with other primers
Amplification from FS_06 to FS_11; 11.6 kb
- Reaction
| what | µl
|
| D. acidovorans DSM-39 | 1
|
| FS_06: (1/10) | 1
|
| FS_11: (1/10) | 1
|
| Phusion Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 6
|
- Conditions
| Biorad MyCycler
|
| Cycles-PCR | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 68 ↓ 0.5 | 5
|
| 72 | 3 min
|
| 18 | 98 | 1
|
| 66 | 5
|
| 72 | 3 min
|
| 1 | 72 | 10 min
|
| 1 | 4 | inf
|
05-07-2013
Amplification from FS_06 to FS_07; 5.2 kb
- Reaction
Gel electrophoresis of DelF-G, Backbone, DelF-G (5.1 kb), DelG (6.4 kb).
| what | µl
|
| D. acidovorans DSM-39 | 1
|
| FS_06: (1/10) | 1
|
| FS_07: (1/10) | 1
|
| Phusion Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 6
|
- Conditions
| Biorad MyCycler
|
| Cycles-PCR | temperature [°C] | Time [s]
|
| 1 | 98 | 5
|
| 14 | 98 | 1
|
| 62 ↓ 0.5 | 5
|
| 72 | 3:20 min
|
| 16 | 98 | 1
|
| 60 | 5
|
| 72 | 3:20 min
|
| 1 | 72 | 12 min
|
| 1 | 4 | inf
|
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