User:Nader A. Khamis/Notebook/Experimental Biological Chemistry AU Fall 2011/2011/09/27

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Objective

  • Synthesis of Gold Nanoparticles using 0.25 mM HAuCl4 and 15 uM BSA in 50 mM Tris Buffer (pH 7.55).
  • Transform DNA from experiment performed on 20th of September into E. Coli cells and then plate the cells on LB/Agar.

Description

Synthesis of BSA Conjugate Gold Nanoparticles

  1. Approximately 0.581 mL of HAuCl4 (0.25mM), 0.974 mL BSA (15uM),and 8.45 mL Tris Buffer pH 7.55 (50 mM) were placed in a test tube.
  2. The small amount of the mixture was pipetted into a cuvette.
  3. The test tube was placed into an oven set at 80°C.
  4. A parafilm was used to cover the cuvette, which contains the sample, and the cuvette was placed into a UV-Vis Spectrophotometer (Shimadzu 2550).
  5. The temperature of the UV-Vis was set at 80°C and a spectra of the sample, which was kept inside the UV-Vis, was taken every 30 minutes for two hours.

Preparation of Agar Plate

  1. Approximately 5g of liquid broth was added to 4g of Agar in 200ml of H2O.
  2. The mixture was heated for 3 minutes in microwave.
  3. Approximately 200ul of 100mg/ml of Ampicilin was added to mixture after cooling to a temperature less than 60°C.

C. DNA Transformation

  1. Approximately 1 uL DpNI Digest was added to the DNA sample.
  2. The sample was placed into a thermocycler for 1 hour at a temperature set at 37°C.
  3. DNA was placed into an ice bath for 15 minutes.
  4. Approximately 5ul of the DNA sample and 50ul of Nova Blue Cells were placed into an eppendorf tube and were mixed.
  5. The mixture was placed on ice for 30 minutes.
  6. The mixture was heat shocked for 30 seconds and was then placed on ice for 5 minutes.
  7. Approximately 250ul of SOC media was pipetted into the mixture and the entire mixture was placed into a shaker for 1 hour at 37 degrees Celsius.
  8. Approximately 200ul of the mixture was pipetted into the plate and was spread.
  9. The plate was placed into a 37°C oven in an inverted position and was stored overnight.

Data

  • The UV-Vis Spectrum was taken at the following times.

T0 - 1:45

T1 - 2:15

T2 - 2:45

T3 - 3:15

T4 - 3:45

  • The graph below is the UV-Vis Spectra of HAuCl4/BSA in Tris buffer (pH=7.5) obtained in the wavelength range of 200-800nm.


Image:Experimental_-_Openwetware_-_September_27th_2011.jpg

  • The graph below exhibits the change in absorbance at 550nm over a period of 2 hours.

Image:Experimental_-_Openwetware_-2-_September_27th_2011.jpg

Notes

  • According to the two graphs, the change in Absorbance was small.
  • According to observations, the solution was clear and no fibers formed.
  • This indicated that no conjugated gold nanoparticles formed.


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