BCH4160/2011:Notebook/Tiffany Byerly Biochemistry Lab

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Contents

Biophysical Chemistry Lab Notebook

  • The purpose of this notebook is to record experiments performed and data collected at High Point University during the Fall 2011 semester

November 15, 2011

With User: Hope Cook

Bomb Calorimetry

  • To gain laboratory experience through the calibration of a bomb calorimeter and the calculation of heat capacity

Procedure

Calibration procedure taken from laboratory manual of User: Jonathan G. Cannon

Data

  • Benzoic acid pellet:
    • mass = 0.984g
    • mw = 122.13 g/mol
    • 6318 cal/g
  • Wire:
    • 23 cal/10cm

Graph

Heat Capacity Calculations

  • Note: All the wire was consumed during the ignition process
    • ΔT = 2.52 °C
    • dH°comb of benzoic acid = -3227 kJ mol-1
    • Total Energy Released = 6239.91 cal --> 26.108kJ
    • Heat Capacity of Bomb Calorimeter (C) = 10.31kJ/°C

November 8, 2011

With User: Hope Cook

Polymerase Chain Reaction

  • To gain laboratory experience in conducting PCR

Procedure

Procedure taken from the Bio-Rad Biotechnology Explorer Crime Scene Investigator PCR Basics Kit

Observations

  • Due to an error, the gel containing the DNA samples was stained before the electrophoresis step. The gel was rinsed thoroughly and was run according to the procedure. However, the sample bands did not appear on the gel. Most likely, the dye interfered with the DNA charge, thus preventing the DNA from being separated. In the future, care will be taken to ensure that electrophoresis is conducted before staining.
  • Even with dilution, the staining dye was extremely concentrated and required multiple overnight rinses.

November 1, 2011

With User: Hope Cook

Enzyme Kinetics Cont.

  • To measure the kinetics of the digestion of starch by cellobiase with and without urea in solution.

Note (Nov. 9, 2011)

For Experiments 1-6, the volume of 1.5% starch should be changed to 1.33mL, not 1.33µL. The amount of buffer should be changed to 0.7mL, not 2mL


Procedure

  • See October 25, 2011 for solution preparation and preliminary data collection
Experiment 1
  1. Add 1.33µL 1.5 % starch solution, 5 µLiodine solution, and 2mL buffer to microcentrifuge tube. Shake to mix
  2. Add 20µL cellobiase solution to microcentrifuge tube. Quickly mix. Place new solution in cuvette and measure absorbance with UV spectrophotometer (See Oct. 25 for parameters).
Experiment 2
  1. Add 1.33µL 1.5 % starch solution, 5 µL iodine solution, and 2mL buffer to microcentrifuge tube. Shake to mix
  2. Add 40µL cellobiase solution to microcentrifuge tube. Quickly mix. Place new solution in cuvette and measure absorbance with UV spectrophotometer.
Experiment 3
  1. Add 1.33µL 1.5 % starch solution, 5 µLiodine solution, and 2mL buffer to microcentrifuge tube. Shake to mix.
  2. Add 0.0555µL of urea to microcentrifuge tube (Will result in 0.5 molal urea). Shake to mix.
  3. Add 20µL cellobiase solution to microcentrifuge tube. Quickly mix. Place new solution in cuvette and measure absorbance with UV spectrophotometer.


Experiment 4
  1. Add 1.33µL 1.5 % starch solution, 5 µLiodine solution, and 2mL buffer to microcentrifuge tube. Shake to mix.
  2. Add 111µL of urea to microcentrifuge tube (Will result in 1 molal urea) . Shake to mix.
  3. Add 20µL cellobiase solution to microcentrifuge tube. Quickly mix. Place new solution in cuvette and measure absorbance with UV spectrophotometer.
  • Note: Depending upon the spectra results, the experimented may be halted here. If the slope of the urea/cellobiase solution appears to be close to zero, do not continue experimenting with higher concentrations of urea is not necessary.
Experiment 5
  1. Add 1.33µL 1.5 % starch solution, 5 µLiodine solution, and 2mL buffer to microcentrifuge tube. Shake to mix.
  2. Add 222µL of urea to microcentrifuge tube (Will result in 2 molal urea) . Shake to mix.
  3. Add 20µL cellobiase solution to microcentrifuge tube. Quickly mix. Place new solution in cuvette and measure absorbance with UV spectrophotometer.
Experiment 6
  1. Add 1.33µL 1.5 % starch solution, 5 µLiodine solution, and 2mL buffer to microcentrifuge tube. Shake to mix.
  2. Add 333µL of urea to microcentrifuge tube (Will result in 3 molal urea) . Shake to mix.
  3. Add 20µL cellobiase solution to microcentrifuge tube. Quickly mix. Place new solution in cuvette and measure absorbance with UV spectrophotometer.

Data

Image:Urea Kinetics Lab Data.xls


Spectra

October 25, 2011

With User: Hope Cook

Enzyme Kinetics

  • To measure the kinetics of the digestion of starch by cellobiase with and without urea in solution.

Solution Preparation

  1. Prepare 1X resuspension buffer (buffer) from original 10X solution (pH = 5)
    • Dissolve 16.7mL original solution in 150mL distilled water
  2. Prepare 1.5% starch solution
    • Dissolve 1.5g starch in 100mL buffer
  3. Prepare 9 molal (mol/kg)urea
    • Dissolve 2.7g urea in 5mL buffer

UV Spectrophotometer Parameters

  • Aquire Mode: "Time Course"
  • Wavelength: 598nm
  • Axes: Absorbance vs. time
  • X-Axis: 0-600s
  • Y-Axis: -0.100-1.00

Procedure

  1. Obtain baseline spectrum of 1.5% solution starch and iodine
    • Dissolve 1.33mL starch solution, 0.7mL buffer, and 5μM iodine in cuvette
    • Measure absorbance with UV spectrophotometer

October 11, 2011

With User: Hope Cook

Binding Fluorescence Cont.

  • To measure the binding fluoresence of a large unilamellar lipid vesicle/peptide solution in order to measure the K of the reaction.

Preliminary Preparations

  1. Rehydrate lipid film (See Oct. 4)by dissolving lipid in sodium phosphate buffer (pH ≈ 7.0).
  2. Agitate the lipid film/buffer solution vigourously for 1 hour (centrifuge, rocker, etc)
    • Mass of lipids = 0.025g
    • MW lipids = 760.10 g/mol
    • 0.0035L buffer
    • Concentration = 9.39mM

Prepare Lipid Vesicles

Controls

  1. Obtain baseline with sodium phosphate buffer
  2. Obtain fluorescence graphs of 2μM, 20μM, and 30μM peptide solution (peptide only) at differing temperatures
    • 15°C, 25°C, and 37°C
    • 1:1 dilution of each concentration in sodium phosphate buffer
      • Results in peptide concentrations of 1μM, 10μM, and 15μM

Experiment

  1. Obtain fluorescence graphs of large unilamellar lipid vesicle/peptide solution

Data

Image:25μM Vesicle Solution in Peptide Solutions + Controls.xls

Spectra

October 4, 2011

With User: Hope Cook

Binding Fluorescence: Preliminary Preparations

  • To prepare peptide solutions of concentrations 2-30μM in preparation for future binding fluorescence lab.
  1. Prepare peptide solution by dissolving 9.6e-4g peptide (MW = 642.79g/mol) in 1 mL sodium phosphate buffer (pH ≈ 7.0)
  2. Measure absorbance of peptide solution using UV spectrophotometer
  3. Measure concentration of peptide solution and Beer's Law
    • Beer's Law: A = ε l c
    • ε = 5,579 M−1 cm−1
    • l = 0.2cm
  4. Prepare three 1 mL dilute peptide solutions in sodium phosphate buffer for analysis (concentrations ranging from 2-30μM)
    • 2μM
    • 10μM
    • 30μM

September 27, 2011

QTi Plots

  • Investigate the graphing program QTi in order to gain proficiency for future labs
  1. Plot an arbitrary data set with multiple Y-value sets
  2. Adjust text, colors, fonts, axis labels, etc
  3. Create custom best fit curve for graph

September 20, 2011

Preparation of Lipid Film

  • To prepare lipid films for use in future experiments


  • Materials (provided by Dr. J. Cannon):
  1. CHCl3
  2. MeOH
  3. Lipid vial
  • Protocol
  1. Make 8mL of 3:1 CHCl3:MeOH (6mL CHCl3/2mL MeOH)
  2. Add 2.5mL of CHCl3:MeOH solution to 1 lipid vial (2 vials total)
  3. Take 1mL of lipid/CHCl3:MeOH solution and place in 4mL vial (5 vials total)
  4. Dry using roto-evaporator
  5. Place vial in desicator overnight to thoroughly dry
  6. Store aliquot in freezer for future use

September 6, 2011

Methyl Red Spectra

  • Objective: To determine the pKa/Ka of methyl red indicator


Protocol

  • See August 30, 2011 for buffer preparation and pH values
  1. Add 150μL methtyl red to 1mL of buffer (pH 4.0) in cuvette, mix
  2. Using spectrophotometer, measure absorbance from 412nm-878nm
  3. Repeat twice
  4. Repeat using buffers at different pHs (see above)
  5. Choose methyl red concentration for spectral measurements
  6. Plot absorbance vs. wavelength
  7. Determine pKa from curve

Data

Image:Methyl Red Spectra.xls


Spectra

August 30, 2011

Buffer Preparation

Protocol

In this experiment, seven sodium phosphate/citrate buffer solutions at pH values ranging from 4.0 to 7.40 were prepared. These buffer solutions were then used to determine the pKa of the pH indicator methyl red through the examination of its absorbance characteristics in the acidic and basic forms as a function of pH.




Sodium Phosphate Buffers (pKa: 6.87)
Theoretical pH Actual pH 0.1M Citric Acid (mL) 0.2M Sodium Phosphate (mL)
3.6 4.0 61.45 38.55
4.2 4.23 58.60 41.40
5.0 5.16 46.40 53.60
5.8 6.0 36.85 63.15
6.6 6.7 27.25 72.75
7.0 7.4 13.05 86.95
7.5 7.16 9.15 90.85
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