User:Asya L. Tucker/Notebook/Asya 571/2015/09/15

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Objective

  1. To prepare gold fibers that we'll will use for the rest of the semester.
  2. To prepare protease samples that we will use for the rest of the semester and record the mass of 15 eppendorf tubes.
  3. To compile the class's UV/Vis, florescence, and average eppendorf tube mass data.
  4. To run our Au fibers with trypsin on the Ocean Optics Spec.

Procedures

  1. Preparing gold fiber
    1. We followed the protocol from September 2nd except we used the stock concentration made by Dr. Hartings today, see description.
  2. Preparing protease samples
    1. We weighed and recorded the mass of an eppendorf tube.
    2. We tared the scale and added between 0.001 g to 0.002 g of protease to the tube.
    3. We labeled the tube with the date, molarity (if 1 mL of water is added), protease (Trypsin), and RAM.
    4. We repeated steps 1-3 for to create 15 protease samples and stored in our freezer box.
    5. Last we uploaded the weights of the 15 eppendorf tubes to dropbox, and averaged all the groups weights.

Ocean Optics Instrument settings - total measurement for 2.5 hours and a reading was recorded ever 2 minutes. Sample preparation

  1. We prepared a 1 mL sample of 52.36 µM of Trypsin and water solution. Then we calculated sample solution we needed to make a 1µM sample in 3 mL.
  2. We added 1 mL of gold fibers, 0.3 mL of buffer, and 0.0573 mL of trypsin after the second reading.
  3. We added 1.643 mL of water to reach 3 mL of sample in the cuvette.

Calculations for volume of trypsin:

    C1·V1 = C2·V2,
     where C1 = 52.36µM
           C2 = 1µM 
           V2 = 3mL
    V1 = 57.3µL

Description

Target Concentrations

Based on the total reaction volume that the class will be using and the final concentrations that will be in each reaction volume, I have determined that the following HAuCl4·H2O and Lysozyme masses are appropriate for today's stock solutions.

Stock solutions

  1. HAuCl4·H2O
    1. 2.5 mM
    2. MW = 339.79 g/mol
    3. Volume = 50 mL
    4. 42.4 mg
  2. Lysozyme
    1. 20 μM
    2. MW = 14307 g/mol
    3. Volume = 100 mL
    4. 28.6 mg

Actual Concentrations

The actual masses are different from the target masses. The concentrations shown below reflect the actual concentrations made for the stock solutions. I used a 50 mL volumetric flask to make the gold stock solution and a 100 mL volumetric flask to make the protein solution. In each case I used HPLC-grade water.

Stock solutions

  1. HAuCl4·H2O
    1. 50.38 mg
    2. MW = 339.79 g/mol
    3. Volume = 50 mL
    4. 2.96 mM
  2. Lysozyme
    1. 28.69 mg
    2. MW = 14307 g/mol
    3. Volume = 100 mL
    4. 20.1 μM

Directions for making samples

Note: The final concentration of gold should be 0.25 mM and the final concentration of lysozyme should be 5.556 μM. This gives a Au:lysozyme ratio of 45, which should yield fibers.

  1. 1 mL samples
    1. Volume of gold stock: 84.4 μL
    2. Volume of protein stock: 276 μL
    3. Volume of water: 640 μL
  2. 5 mL samples
    1. Volume of gold stock: 422 μL
    2. Volume of protein stock: 1,380 μL
    3. Volume of water: 3,198 μL


Data

  • The table below lists the protease samples we prepared and the mass of the corresponding eppendorf tube.
Mass of Eppy Tube (g) Mass of Protease (g) Concentraton (micromolar)
1.026910.0010846.35
1.026220.0014863.52
1.02240.0011750.21
1.019440.0016972.33
1.029220.0018177.68
1.026250.0012854.93
1.019090.0016570.82
1.020360.0018278.11
1.019220.0012854.93
1.020240.0012252.36
1.020970.0015466.09
1.024220.0017575.11
1.026690.0012453.22
1.029360.0014863.52
1.029230.0015566.52
  • The table below lists the the class's eppendof tube masses (my groups data set are under 'RAM 9/15').
AMS 9/9 AMS 9/15 LMN 9/9 JBN 9/9 MB 9/9 RAM 9/15 AMS 9/16
1.021571.031071.031041.0151.0261.030051.03028
1.031621.031921.028591.013481.0261.02551.02355
1.027981.026971.01991.01791.0261.026761.02919
1.032751.019231.027531.029541.0261.028691.02559
1.030571.029981.016461.02281.0261.01656
1.013921.030591.030781.020481.0261.01995
1.029381.020251.024191.019451.0261.01707
1.029171.029321.013081.027231.0261.02417
1.029931.033311.028961.029511.0261.02518
1.022231.02871.02881.015171.0261.01762
1.020551.025541.026021.029541.0261.0349
1.031291.013721.024561.032111.0261.03024
1.013591.018531.017191.01921.0261.02016
1.029971.020511.028631.027811.0261.0205
1.031171.030151.02851.0261.028121.01972
Average Mass (g) 1.025135426 Standard Deviation 0.005373647
  • The table below is the raw data for the graph following it. The graph is a concentration vs absorbance of the class's compiled UV/Vis data at 280 nm.
Group Concentration A(280nm)
Group 120.052
50.16
80.239
100.292
150.444
Group 30.78750.026
2.6250.091
5.250.186
7.50.252
150.502
Group 40.9650.032
1.930.069
3.860.123
7.2250.261
15.4450.517
Group 51.560.052
3.130.108
6.250.221
12.50.441
150.54


  • Ocean Optics graphs are below


Results

  1. We did not create any gold nanoparticles or nanofibers.
  2. The average mass of an eppendorf tube is 1.025135426 and the standard deviation was 0.005373647.
  3. As a class the Lysozyme Molar Absorbtivity Coefficient: 33200 M-1 cm-1 and an R2 equal to 0.9846.



Matt HartingsYou need to discuss the results from #1, #2, and #3 from your objectives. What is the data from the tube measurement? What was the result of your measurements with the ocean optics? What were the results of your fiber synthesis?

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