IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/08/06: Difference between revisions
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==Getting Promoter Plasmid and Iron Assay with Fur- Mutants== | ==Getting Promoter Plasmid and Iron Assay with Fur- Mutants== | ||
=== 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 === | === 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 | |||
{| border="1" cellpadding="2" | {| border="1" cellpadding="2" | ||
|+Protocol for | |+Protocol for Varying Iron Concentration in Medium | ||
|- | |- | ||
! [Fe]/μM !! 0 !! 4 !! 8 !! 12 !! 16 !! 20 | ! [Fe]/μM !! 0 !! 4 !! 8 !! 12 !! 16 !! 20 | ||
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* 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 | |||
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Revision as of 04:48, 7 August 2008
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Getting Promoter Plasmid and Iron Assay with Fur- MutantsPromoter Plasmid
Iron Assay with Fur- Mutants
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