BCH4160/2011:Notebook/Hope Cook

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8-30-11

Buffer preparation Procedure: Seven phosphate/ citrate buffer solutions were prepared, with pH values ranging from 4.0 to 7.40. The buffers solutions were used to determine the pKa of methyl red.

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

9-27-11

Purpose: The acidic and basic forms of methyl red were examined to determine optimal volumes of methyl red stock solution and buffer to use in each pH assay.

Procedure:

  1. Add 150 microliters of methyl red to 1mL of buffer in cuvette and mix,
  2. Use spectrophotometer to measure absorbance from 412nm-878nm, repeat two times,
  3. repeat using buffers at different pHs,
  4. choose methyl red concentration for spectral measurements,
  5. plot absorbance vs. wavelength, and determine pka from the curve

QTi Plots(Data is in with buffer data)

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

Buffer Data

Image:Buffer data .xls

Preparation of Lipid Film

objective: to prepare lipid films for use in future labs. Procedure:

  1. Make 8mL of a 3:1 CHCl3:MeOH solution this is done by using 6mL of CHCl3 and 2mL of MeOH
  2. Add 2.5 mL of CHCl3:MeOH solution to a lipid vial
  3. Place 1mL of lipid/CHCl3:MeOH solution in a 4mL vial
  4. Roto evaporate
  5. Put vial is desicator overnight
  6. Store lipid film in the freezer


Fluorescence

With User: Tiffany Byerly

Objective: To prepare peptide solutions of concentrations 2-30 micromolar. To measure th binding fluoresence of a large unilamellar lipid vesicle/peptide solution in order to measure the K of the reaction.

Procedure: October 4

  1. Prepare peptide solution by dissolving 9.6e-4g peptide of MW=6.42.79 g/mol in 1mL sodium phosphate buffer of pH 7.
  2. Measure absorbance of peptide solution with UV spectrophotometer
  3. Measure concentration of peptide solution and use Beer's Law
    A=Elc
    E= 5,579M-1 cm-1
    l-0.2cm
  1. Prepare three 1mL dilute peptide solutions in sodium phosphate buffer for analysis (concentrations ranging from 2-30 micromolar)
  We used 2, 10, and 30 

Procedure: October 11

  1. Rehydrate lipid film by dissolving lipid in 3.5 ml of sodium phosphate buffer
  2. Vortex the lipid film/buffer solution for 1 hour

Procedure for preparing lipid vesicles was found from:

Controls

  1. Take baseline with sodium phosphate buffer
  2. Obtain fluorescence graphs of 2,20, and 30 micromolar peptide solution at different temperatures.
  we used 15, 25, and 37 degrees Celcius, a 1;1 dilutions of each concentration in buffer, and this resulted in 1, 10, 15 micromolar peptide concentrations

Experiment

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

Data

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

Enzyme Kinetics

With With User:Tiffany Byerly

Objective: To measure the kinetics of the digestion of starch by cellobiase with and without urea


Procedure October 25 Solution Preparation

  1. Prepare 1X resuspension buffer from 10X solution (pH = 5)

Dissolve 16.7mL original solution in 150mL distilled water

  1. Prepare 1.5% starch solution

Dissolve 1.5g starch in 100mL buffer

  1. Prepare 9 molal (mol/kg)urea

Dissolve 2.7g urea in 5mL buffer

  1. Set parameters for UV spectrophotometer: Aquire Mode: "Time Course", Wavelength: 598nm, Axes: Absorbance vs. time, X-Axis: 0-600s, Y-Axis: -0.100-1.00
  2. Obtain baseline spectrum of 1.5% solution starch and iodine
  3. Dissolve 1.33 mL starch solution, 0.7mL buffer, and 5μL iodine in cuvette
  4. Measure absorbance with UV spectrophotometer

Procedure November 1 Experiment 1

  1. Add 1.33 mL of 1.5% starch solution, 5μL of iodine, and 0.7mL of buffer to a microcentrifuge tube. Shake to mix.
  2. Add 20 μL of cellobiase solution to the microcentrifuge tube. Quickly mix. Put solution in cuvette and measure the absorbance using UV spectrophotometer.

Experiment 2

  1. Add 1.33 mL of 1.5% starch solution, 5μL of iodine, and 0.7mL of buffer to a microcentrifuge tube. Shake to mix.
  2. Add 40 μL of cellobiase solution to the microcentrifuge tube. Quickly mix. Put solution in cuvette and measure the absorbance using UV spectrophotometer.

Experiment 3

  1. Add 1.33 mL of 1.5% starch solution, 5μL of iodine, and 0.7mL of buffer to a microcentrifuge tube. Shake to mix.
  2. Add 0.0555μL of urea to the microcentrifuge tude, results in 0.5molal urea. Shake to mix.
  3. Add 20 μL of cellobiase solution to the microcentrifuge tube. Quickly mix. Put solution in cuvette and measure the absorbance using UV spectrophotometer.

Experiment 4

  1. Add 1.33 mL of 1.5% starch solution, 5μL of iodine, and 0.7mL of buffer to a microcentrifuge tube. Shake to mix.
  2. Add 111 μL of urea to the microcentrifuge tube, results in 1 molal urea. Shake to mix.
  3. Add 20 μL of cellobiase solution to the microcentrifuge tube. Quickly mix. Put solution in cuvette and measure the absorbance using UV spectrophotometer.
Note: Depending on the spectra, the experiment may be stopped here. If the slope of the urea/ cellobiase solutions appears to be close to zero, do not continue experimenting with higher concentrations of urea. 

Experiment 5

  1. Add 1.33 mL of 1.5% starch solution, 5μL of iodine, and 0.7mL of buffer to a microcentrifuge tube. Shake to mix.
  2. Add 222μL of urea to the microcentrifuge tube, results in 2 molal urea. Shake to mix.
  3. Add 20 μL of cellobiase solution to the microcentrifuge tube. Quickly mix. Put solution in cuvette and measure the absorbance using UV spectrophotometer.

Experiment 6

  1. Add 1.33 mL of 1.5% starch solution, 5μL of iodine, and 0.7mL of buffer to a microcentrifuge tube. Shake to mix.
  2. Add 333μL of urea to the microcentrifuge tube, results in 3 molal urea. Shake to mix.
  3. Add 20 μL of cellobiase solution to the microcentrifuge tube. Quickly mix. Put solution in cuvette and measure the absorbance using UV spectrophotometer.

Data

Image:Urea Kinetics Lab Data.xls

Polymerase Chain Reaction

With User:Tiffany Byerly

Objective: To gain laboratory experience in conducting PCR

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

Observations:

  1. Due to 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, therefore preventing the DNA from being separated. In the futue, care will be taken to ensure that electrophoresis is conducted before staining the gel.
  2. Even with dilution, the staining dye was extremely concentrated and required multiple overnight rinses.

Bomb Calorimetry

With User:Tiffany Byerly

Objective: To gain laboratory experience throught the calibration of a bomb calorimeter and the calculation of heat capacity.

Procedure: taken from the laboratory manuel of User: Jonathan G. Cannon

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


Heat Capacity Calculations Note: All the wire was consumed during the ignition process

change in temperature= 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= 10.31kJ/°C

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