User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/09/28

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Objective

To repeat gold nanoparticle synthesis and to continue protein expression started on 09/27/11.

Description

1. Nanoparticle Synthesis

  1. Combine 10 mL of water with 0.974 mL of 0.0015 mM BSA and 0.581 mL of 0.25 mM HAuCl4 in a capped test tube at room temperature.
  2. Take UV vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min.
  3. Keep cuvette in the UV-Vis instrument at 70°C between spectra.
  4. After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.

2. Protein Expression II

  1. Centrifuge the starter cultures at 4500 rpm for 15 min
  2. Re-suspend the cell pellets in 4mL of fresh LB.
  3. Inoculate the expression culture media by dividing the resuspended cells among the Fernbach flasks.
  4. Incubate the expression cultures at 37C and 160rpm until the absorbance of the media at 600 nm = 0.6.
  5. Add 1 mL of 0.4 M IPTG to each flask in order to induce protein expression
  6. Continue shaking for 3-4 hr
  7. Harvest the cells by centrifuging at 4500 rpm for 15 min at 4°C with a SH-3000(BK) rotor.
  8. Collect the cells in tris buffer (pH 9) and place in the -80°C freezer

Data

The absorbance values for the AuNP solution were corrected by subtracting the absorbance values for a blank acetate buffer. The corrected absorbance values at 550 nm were graphed against time. The graph shows a linear relationship after 60 min, indicating that AuNPs were formed in solution.

Absorbance vs time 9-28-11.jpg

This graphs shows the corrected absorbance against wavelength. Absorbance data collected at 150 min from 200 nm-300 nm was ignored, because of outside interference with the UV-vis instrument.

Absorbance vs wavelength 9-28-11.jpg

A magnified version of this graph shows that absorbance values at around 550 nm increased over time, indicating the creation of AuNPs.

Absorbance vs wavelength magnified 9-28-11.jpg

Notes