User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/25

Objective

• to make 4, 1L LB solutions in Fernbach flasks and 4, 35mL LB solutions in 250mL flasks,
• to autoclave them for 1.5 hours, and
• to add cells and antibiotics to the solutions.

In addition,

• to remake luminol and HRP solutions, and
• to rerun fluorescence at 510nm on the luminol, hydrogen peroxide, phenol, and HRP mixture.

Description

*Preparing Starter Culture Media

• • 0.8752g, 0.8754g, 0.8751g, and 0.8756g of LB were added to four separate 250mL Erlenmeyer flasks.
  • 35.0mL H₂O was added to each flask.
  • The flasks were covered with foil and labeled.

*Preparing Expression Culture Media

• • 25.0015g, 25.0013g, 24.9991g, and 24.9998g of LB were added to four separate Fernbach flasks.
  • 1.0L H₂O was added to each flask.
  • The flasks were covered with foil and labeled A, B, C, and D, in order to distinguish each flask.

*Autoclaving

• • The power was turned on
  • Water was poured into the chamber manually because the water pipe was clogged.
  • The black knob was set to STE (sterilize)
  • 2 Fernbach and 2 Erlenmeyer flasks were loaded into the chamber.
  • The autoclave door was closed and sealed shut.
  • The temperature was set to about 121°F
  • The autoclave was set to run for an hour.
  • After the hour had elapsed, the flasks were left to sit in the autoclave for 30 minutes so the pressure in the autoclave could return to atmospheric pressure.
  • The black know was turned to off
  • Gloves were used to unload the sterilized material.

*Preparing Luminol Solution

• • The same protocol as last week was used to prepare the luminol solution.
  • 0.00443g of luminol was measured out, so the calculations for actual concentration from last week are accurate.
  • Actual Concentration: 2.5 × 10-3 M in H₂O.
  • 0.0800 grams of Sodium Carbonate was added to the luminol solution so that the luminol would dissolve in the solution.
  • 0.4800 grams of sodium bicarbonate was added to the solution to adjust the pH.

*Preparing HRP Solution

• 1mg of HRP was added to 1mL water, creating a 23μM HRP solution.
• The HRP was further diluted to 2.3μM by adding 1mL HRP solution to 9mL H₂O.

*Running Fluorescence

• The fluorometer was set to have an excitation at 350nm (with a 15nm slit) and emission at 430nm (with a 20nm slit).
  • The source was turned off because in chemiluminescence, the source is the sample itself.
• The 13.2μL Phenol, 920μL Luminol, 990μL hydrogen peroxide, and 66μL HRP were added (the HRP was added at the last second) with the following concentrations:

Data

• Please refer to Melissa's entry for the results of the HRP Chemiluminescence Assay, including concentrations of each trial and a graph of intensity of phenol-enhanced solutions of luminol and hydrogen peroxide with HRP.

• • The HRP solution (23μM) was diluted to 2.3μM, 1.15μM, 0.23μM, and 23nM via serial dilution.
  • The data obtained from the 2.3μM HRP:425μM Hydrogen Peroxide, 2.3μM HRP:1.7mM Hydrogen Peroxide, and 1.15μM HRP:1.7mM Hydrogen Peroxide (all containing 18mM phenol and 1.25mM Luminol) indicate that the reaction is occurring too quickly with the current HRP concentrations, as the slope of each of the lines created are too steep for effective analysis. The concentration of the HRP needs to be significantly decreased in order to obtain a clear curve.