User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/09/27

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Contents

Objective

  • Perform nano-particle synthesis using .25 mM Chloroauric Acid(HAuCl4) and 15 μM Bovine Serum Albumin(BSA) in 50 mM Tris Buffer pH 7.55.
  • Transform DNA from experiment performed on 9/20 into E. Coli cells and then plate the cells on LB/Agar media.

Description

  • A. Nanoparticle Synthesis Procedure:
  • Mixed .581 mL of HAuCl4 (.25mM) with 0.974 mL BSA (15μM)and 8.45 mL Tris Buffer pH 7.55 (50 mM) in test tube
  • Small volume of mixture was transferred to cuvette which was then covered with parafilm, and placed into UV-Vis Spectrophotometer (Shimadzu 2550)
  • UV-Vis was set at 80 Degrees Celsius, and spectra (200 - 800 nm) of the cuvette were taken every 30 mins for 2 hours
  • Mixture in test tube was placed into oven set at 80 Degrees Celsius


  • B. Agar Plate Procedure:
  • 5 g LB was mixed with 4 g Agar in 200 mL H2O
  • Mixture was heated in microwave for 3 minutes
  • 200 μL of Ampicillin was added to mixture
  • Mixture was then poured into petri dish to harden


  • C. DNA Transformation Procedure:
  • 1 μL DpNI Digest was added to DNA sample
  • DNA sample was then placed into thermocycler set at 37 Degrees Celsius for 1 hour
  • After shaking, DNA sample was placed in ice bath for 15 minutes
  • 5 μL of DNA sample was mixed with 40 uL Nova Blue E. Coli cells in new eppendorf tube
  • DNA/Cell Mixture was placed on ice for 30 minutes, heat shocked for 30 seconds, and then placed on ice for 5 minutes
  • 250 μL of SOC media was added to mixture
  • Mixture was then placed into shaker for 1 hour at 37 degrees Celsius
  • 100 μL of mixture was spread on plate
  • Plate was stored(inverted) in oven at 37 degrees Celsius overnight

Data

  • Full spectrum of mixture.

Image:OC-9-27-1.jpg


  • Spectrum focused at 550 nm. Very indiscernible curve is visible.

Image:OC-9-27-2.jpg



  • Change in absorbance at 550 nm over two hours.

Image:OC-9-27-3.jpg

Notes

  • Molar ratio of Au/BSA = 9.92
  • Solution remained clear after 2 hours in oven.
  • The slope on the graph of absorbance vs. time at 550 is near zero which indicating that gold nanoparticles were not formed.
  • Compared to previous results, Tris buffer is not efficient for nanoparticle synthesis. Using H2O buffer yielded the best results, therefore future experiments will have H2O buffer.
  • Tris buffer: tris(hydroxymethyl)aminomethane (HOCH2)3CNH2. Tris buffer is commonly used in experiments involving amines.
  • LB/Agar - Lysogeny broth nutrient agar commonly used for cultivation of bacteria.
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