From 2013.igem.org
26-07-2013
Restriction digest of FS_02 to FS_05; 11.2 kb; 04-07-2013 with EcoRI-HF
Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)
Incubation at 37°C for 1hour 45min
| what | µL
|
| FS_02 to FS_05 (04-07-2013) | 15
|
| EcoRI-HF | 0.5
|
| Buffer CutSmart | 2
|
| dd H2O | 2
|
| Expected fragment lengths [bp] | 4624, 4354, 2260
|
Results:
- restriction digest did not work as expected
- test restriction will be repeat with higher amount of enzyme and longer incubation time
27-07-2013
Amplification of FS_02 to FS_05; 11.2 kb
Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)
- Reaction
| what | µL
|
| D. acidovorans DSM-39 | 1
|
| FS_02: (1/10) | 2.5
|
| FS_05: (1/10) | 2.5
|
| Phusion flash Master Mix | 25
|
| DMSO | 2.5
|
| dd H2O | 16.5
|
- Conditions
| Biorad MyCycler*
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 68 ↓ 0,5 | 5
|
| 72 | 3:00
|
| 18 | 98 | 1
|
| 66 | 5
|
| 72 | 3:00
|
| 1 | 72 | 10 min
|
| 1 | 4 | inf
|
Results:
- Amplification of DelAE worked, but a slight smear as well as several unexpected bands occured
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit but PCR has to be further optimized in order to improve product quality
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