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Team:Heidelberg/Templates/Del week13 AF
From 2013.igem.org
Contents |
26-07-2013
Restriction digest of FS_02 to FS_05; 11.2 kb; 04-07-2013 with EcoRI-HF
File:20130726 2,5µllog2 AEEcoRIHF AFEcoRIHF GBGlII OPEcoRIHFbesch.png
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) | 15 |
| EcoRI-HF | 0.5 |
| Buffer CutSmart | 2 |
| dd H2O | 2 |
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
File:20130727 2log 2xAE 2xAF 1kb 2xIliabesch.png
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)
File:20130727 2log 2xAE 2xAF 1kb 2xIliacut.png
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 | 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
