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Team:Panama/9 August 2010
From 2010.igem.org
August 9
More electrophresis
High resolution 2% agarose gel (for small fragments). In this electrophoresis we used two (1 and 5) molecular weight markers, the Lamda DNA/Hind III marker from Biotools. We decided to load duplicates of the samples, so we have the Promoter and RBS on lanes 2 & 3; lane 4 is empty and once again, the Promoter and RBS are in lanes 6 & 7.
After the electrophoresis was done, we proceed to stain the gel with ethydium bromide: Ethydium Bromide staining procedure:
The stock concentration was at 10mg/ml.
The concentration for gel staining was 0.5ug/ml.
We used 15ul of Ethydium Bromide in 300ml of dH2O. We stained it covered for 30 minutes. Afterwards, we washed it in water for 15 minutes each time.
We weren't able to visualize the band of the small fragments of the digestion. We can only see the band that corresponds to the plasmid. It is possible that the intensity of the small bands is not enough for them to be seen.
Restriction reaction Repetitions:
1. Promoter (using NEB restriction enzymes)
2. Promoter (using other restriction enzymes)
3. RBS
The reason behind testing different batches of the same restriction enzymes for the promoter was to find out the reason why we didn't see anything when we ran electroforesis gels. We had thought that maybe the restriction enzymes were not doing their job, so we tried digesting the parts with enzymes that INDICASAT provided.
PsB1C3 Plasmid transformation (Stock):
- LB plates with Cloranfenicol at a concentration of 33ug/ml.
- LB plates with Tetracycline at a concentration of 15ug/ml.
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