IGEM:Groningen/Notebook/iGEM 2011/2011/08/04

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Plates of PBAD-RBS-GFP-DT (overnight ligation sample) look very nice! Self ligation control does not contain much colonies.
PBAD- RBS-GFP-DT contains more than ten fold colonies compared to the self ligation plates...
Colony PCR of PBAD-RBS-GFP-DT (sample FB)
Composition master mix:
10× Taq buffer: 40μl
dNTPs 10mM: 8μl
MgCl2: 24μl
Forward biobrickvector primer: 8μl
Reverse biobrickvector primer: 8μl
Taq polymerase: 2μl
MQ water: 310μl

PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extenstion: 72°C for 30s
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

Overnight colonies of PhybB-RBS-GFP-DT and pGFP-rrnB were grown in minimal medium again for logfase for the flow cytometry
At 14:30 start Flow cytometry:
Protocol of Maarten Mols:
Media:File Flow Cytometry of Maarten Mols.ogg

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