User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/25

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Purpose

  • To start culture for cell growth with transformed ADA strand cells, amplification of cells eventually leads to purification of more ADA proteins
  • Continue with HRP assay measurements using florometer in order to determine the concentration reactants needed for best HRP assay reactivity

Procedure

  • Four LB solution for starter culture was made by mixing 0.875g of LB in 35mL of distilled water in 250mL Erlenmeyer flasks. Four of 1L LB solution for cell amplification 250g with 1L distilled water in 2.8L Erlenmeyer flasks. Broth were autoclaved in liquid cycle.
  • Abigail E. Miller 17:46, 7 October 2012 (EDT): did you put exactly 0.875 g and 250 g (shoudl be 25 g)? what is liquid cycle and how did the autoclave work?
  • New horseradish peroxidase were made by mixing 1mg of horseradish peroxidase in 1mL of distilled water. The calculated final concentration of horseradish peroxidase is 2.3uM. The enzyme were diluted ten folds to 230nM and 23nM to test luminol and H2O2 reaction.
  • New 4-Iodophenol was made with water and sodium bicarbonate. See Melissa's Notebook for details on making the 4-Iodophenol stock solution.
  • One colony from ADA transformation cell plate were taken and dipped into 4mL starter LB culture. 4uL of 0.4M ampicillin were added to starter. Cell culture were placed in shaker overnight and for 15 hours overnight under 37C.
  • Abigail E. Miller 17:46, 7 October 2012 (EDT):you did not use ampicillin you used kanamycin. what speed did it shake at?

Notes

  • Please see Melissa's Notebook for data obtained from luminol-H2O2 reaction with HRP assay. The results found from luminol-H2O2 reaction remained inconclusive, and more trials will be run with different ratios of luminol and H2O2, and more concentrations of HRP would be used to see the effect of enzymatic reactions.
  • Ampicillin were added in order to make sure the cells growing in LB culture were strictly cells containing ADA transforming genes.
  • Abigail E. Miller 17:47, 7 October 2012 (EDT):the data shoudl be in your notebook. and again not ampicillin, you used kanamycin.


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