IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/08/06: Difference between revisions

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* Plastic ware used throughout to prevent alteration of iron concentration
* Plastic ware used throughout to prevent alteration of iron concentration
* pH paper used to test iron stock to be around pH7
* pH paper used to test iron stock to be around pH7
 
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|+Protocol for Varying Iron Concentration in Medium
|+Protocol for Varying Iron Concentration in Medium
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* Initial OD Reading 30min after addition of Kanamycin and right before incubation = 0.197
* Initial OD Reading 30min after addition of Kanamycin and right before incubation = 0.197

Revision as of 04:47, 7 August 2008

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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

  • 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

<html><br> {| border="1" cellpadding="2" |+Protocol for Varying Iron Concentration in Medium |- ! [Fe]/μM !! 0 !! 4 !! 8 !! 12 !! 16 !! 20 |- ! Vol. of Fe stock/ml | 0 || 0.01 || 0.02 || 0.03 || 0.04 || 0.05 |- ! Vol. of SDW/ml | 0.50 || 0.49 || 0.48 || 0.47 || 0.46 || 0.45 |- ! Vol. of fur- cells in M9/ml | 9.5 || 9.5 || 9.5 || 9.5 || 9.5 || 9.5 |- |} <br></br></html>

  • 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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