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.

*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 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.