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

From OpenWetWare

Jump to: navigation, search
Experimental Biological Chemistry I Main project page
Previous entry      Next entry

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.
  • For the liquid cycle, broth was sterilzed for 35 minutes. After 35 minutes of sterilization, the pressure were waited until turned to 0. LB solutions were unloaded and taken out.
  • 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 kanamycin were added to starter. Cell culture were placed in shaker at 225rpm and for 15 hours overnight under 37C.

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.
  • Kanamycin were added in order to make sure the cells growing in LB culture were strictly cells containing ADA transforming genes.


Personal tools