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Team:Bielefeld-Germany/Labjournal/June
From 2013.igem.org
June
Milesstones
1.Week
MFC
Mediators
Cytochromes
Biosafety
Porines
2.Week
MFC
Mediators
Cytochromes
Biosafety
Porines
3.Week
MFC
Mediators
| Reagent | 1x | 8x |
|---|---|---|
| Hf-Buffer 5x | 10.0 | 88.0 |
| dNTPs (10mM) | 2.0 | 17.6 |
| Primer1 (10mM) | 1.0 | 8.8 |
| Primer2 (10mM) | 1.0 | 8.8 |
| Template (16.5ng) | 3.0 | 26.4 |
| DMSO | 4.0 | 35.2 |
| Phusion (0.5u) | 1.0 | 8.8 |
| H2O | 26.0 | 228.8 |
| Program Phusion | ||||
|---|---|---|---|---|
| 98 °C | 98 °C | 68 °C | 72 °C | 72 °C |
| 30s | 10s | 30s | 1:35 | 6:00 |
| 35 cycles | ||||
|
Notes:
Size: 1800bp Primer1: Primer2: | ||||
Cytochromes
Cultivation of Shewanella oneidensis MR-1 in liquid LB medium at 30 °C
Isolation of genomic DNA from S. oneidensis
4-2006-451: Conc.:453.1 ng/µl OD260/280: 1.92 OD260/230:2.78
4-2006-452: Conc.:447.1 ng/µl OD260/280: 1.92 OD260/230:2.67
Dilution to PCR template: 4-2006-453: 5.5ng/µl
Amplification of the mtrCAB cluster with gradient PhusionPCR
Gradient: 55.8-56.7-57.8-59.1-60.4-61.7-62.9-63.9
Clear Bands at the expected 5.2kb
PCR-CleanUp of lane 2 and 5 with the Machery-Nagel-PCR-CleanUp-Kit
Lane2: 4-2106-451: 7.4 ng/µl
Lane5: 4-2106-451: 8.5 ng/µl
