User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/09/28

From OpenWetWare

Jump to: navigation, search

Customize your entry pages

Objective

To continue protein expression from the previous day and to synthesize AuNP nanoparticles.

Procedure

AuNP synthesis:

  1. Combine 10 mL of 50 mM acetate buffer (pH 5.46) 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. Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions.
  4. After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.


Protein Expression (continued):

  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


Results

Water as a Buffer:



Each time interval had a positive effect on the absorbance of the cuvette solution. This suggests that AUNPs were being formed as time increased. A slight disturbance during t=150 occurred such that values from wavelengths 200-300 were removed from the data set and marked as invalid.

The positive linear relationship suggests that AUNP were formed in the solution as time increased.


Notes


Personal tools