IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/08/06

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Promoter Plasmid

 * No colonies formed on the Kanamycin plates for I0500 and pUC control still
 * Re-selected promoter plasmid R0010 with ampicillin resistance in well 5E on plate 1002
 * Extracted R0010 from filter paper following standard protocol
 * Transform competent TOP10 with R0010 and J63006 again following standard transformation protocol with some amendments
 * R0010: 5μL DNA + 25μL TOP10 + 55μL SOC
 * J63006: 15μL DNA + 25μL TOP10 + 45μL SOC
 * Plate out neat and 1/10 dilution on freshly prepared ampicillin plates of 100μg/mL
 * Incubate overnight at 37°C

Iron Assay with Fur- Mutants

 * Added 0.222g of iron sulphate into 400mM sodium citrate stock prepared beforehand
 * Notice that some iron sulphate solid cannot dissolve into solution completely which may affect iron concentration slightly
 * Plastic ware used throughout to prevent alteration of iron concentration
 * pH paper used to test iron stock to be around pH7


 * Initial OD Reading 30min after addition of Kanamycin and right before incubation = 0.197
 * Cell Density can be obtained from graph :http://openwetware.org/wiki/IGEM:Cambridge/2008/Notebook/Voltage/OD600_Calibration


 * Placed plastic tubes with cells and medium inside the plastic jar borrowed from the Path Dept
 * Add SDW into package containing chemical reagents and catalyst to activate it
 * Package produces hydrogen gas which reacts with oxygen in air in the presence of palladium catalyst
 * Carbon dioxide also produced to form a CO2-enriched environment, favouring anaerobic growth by E.coli
 * Cultures incubated at 37°C for 2 nights