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Team:Heidelberg/Templates/Del week13 EG
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(Created page with "==28-07-2013== ===Amplification from FS_04 to FS_09 ; 14.4 kb=== 2 reactions with conditions I and II :'''Reaction''' {| class="wikitable" |- ! what !! µl |- | ''D. acidov...")
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(Created page with "==28-07-2013== ===Amplification from FS_04 to FS_09 ; 14.4 kb=== 2 reactions with conditions I and II :'''Reaction''' {| class="wikitable" |- ! what !! µl |- | ''D. acidov...")
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Revision as of 00:39, 30 September 2013
Contents |
28-07-2013
Amplification from FS_04 to FS_09 ; 14.4 kb
2 reactions with conditions I and II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 2 |
| FS_09: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 4:40 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
- Conditions II
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 4:40 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 4:40 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
Amplification from FS_26 to FS_07
_FS7to26(68)_FS24to26(65)_FS24to26(68).png)
This amplification did not make sense, two reverse Primer were used. We mixed up Primer FS_24 with Primer FS_26.
2 reactions with conditions I and II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_26: (1/10) | 2 |
| FS_07: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
- Conditions II
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification of DelEG did not work, neither with annealing at a constant temperature of 65°C nor with a touchdown starting from 68°C
- later on it was discovered, that primers had been mixed up
Amplification from FS_26 to FS_24
This amplification did not make sense, two reverse Primer were used.
File:20130729 log2 FS21toFS24 FS7to26(65) FS7to26(68) FS24to26(65) FS24to26(68).png
2log ladder / FS21-FS24 60const / FS07-FS26 65const / FS07-FS26 68const / FS24-FS26 65const / FS24-FS26 68const; run at 100 V, 0.8 % gel (TAE)
2 reactions with conditions I and II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_26: (1/10) | 2 |
| FS_24: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
- Conditions II
| Biorad C1000 Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification of DelEG did not work, neither with annealing at a constant temperature of 65°C nor with a touchdown starting from 68°C
- later on it was discovered, that primers had been mixed up
_FS7to26(68)_FS24to26(65)_FS24to26(68).png){kind=link}