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Team:Heidelberg/Templates/Del week10 AF
From 2013.igem.org
Contents |
03-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
File:20130703 DelAF DelFG.png
Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)
File:20130703 DelAF DelFG cut.png
Gel after removing the DelA-F-fragment, 03-07-13.
- Reaction
| µL 1st PCR | what | µL 2nd PCR |
|---|---|---|
| 1 | D. acidovorans | 1 |
| 2.5 | FS_02 (1/10) | 2.5 |
| 2.5 | FS_05 (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 |
| 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 DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- sample has to be purified before further use since contamination with propanol was present
04-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
File:20130704 DelAF DelFGnew.png
Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013
File:20130704 DelAF DelFG cutnew.png
Gel after removing DelA-F-fragment, 04-07-2013
- Reaction
| what | µL 2nd PCR |
|---|---|
| D. acidovorans | 1 |
| FS_02 (1/10) | 2.5 |
| FS_05 (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 DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
- PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
