User:Benjamin Friedel/Notebook/CHEM 471/2015/09/16

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Lysozyme AuNP Fiber Reaction and Buffer Making

Objective

Today 1ml reactions were redone to form AuNP fibers with Lysozyme as reactions from 09/15/2015. Additionally, 500 of mL of 10mM Tris buffer at pH9 and 500mL of 10mM phosphate buffer at pH7 were made.

Protocol

  1. First, 16x 1mL Lysozyme AuNP fiber reactions were set up as reactions from http://openwetware.org/wiki/User:Benjamin_Friedel/Notebook/CHEM_471/2015/09/15 [09/16/15] were unsuccessful.
    1. Using the same procedures as on 09/02/2015 AuNP fiber reaction mixes were set up with 1 mL samples. Below are the contents and volumes of each reaction mix with 16x1mL samples and 10x5mL samples. All reaction mixes were prepared at room temperature with stock solutions prepared by Dr. Hartings previously the same day (09/16/2015). Reactions from 09/15/15 failed possibly because stock solutions were made in incorrect concentrations. Samples were covered in aluminum foil then heated in oven by Dr. Hartings for 4 hours at 80˚C. The reaction cycle also included 30 minutes to heat from room temperature to 80˚C and 30 minutes to return to room temperature before and after the 4 hour reaction. This is the second attempt at AuNP synthesis in 5mL reaction mix volumes, as the first attempt on 09/02/2015 failed (for both the 1mL and 5mL samples).

NOTE: Determine fate of samples after reaction.

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
  1. Then buffer solutions were made in the below specifications
    1. 500 mL of 10 mM Tris buffer, pH 9
      1. The Science Gateway buffer calculator was used for the Tris buffer. Using the above volume, molarity and pH and selecting Tris as the buffer the mass of Tris to be used was calculated to be 0.6057g. The real mass used was 0.6079g. This was added to 500mL of HPLC water in a 500mL volumetric flask.
        1. pH was measured with a pH meter and adjusted with 1M HCl until pH was reduced to 9. Conductivity was measured to be 281.2uS/cm with a conductivity meter.
  1. 500mL of 10mM phosphate buffer at pH 7.
    1. The Clymer phosphate buffer calculator was used for the phosphate buffer to calculate the amount of dibasic and monobasic sodium phosphate used using the above volume, molarity and pH.
      1. Sodium phosphate (dibasic)= 0.50259g Sodium phosphate (monobasic=0.29185g
        1. The conductivity measured 1655uS/cm and pH=7

buffers were verified for pH and conductivity by Dr. Hartings and then disposed of in the sink.