Objective
To continue protein expression from the previous day and to synthesize AuNP nanoparticles.
Procedure
AuNP synthesis:
- 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.
- Take UV-vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min.
- Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions.
- After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.
Protein Expression (continued):
- Centrifuge the starter cultures at 4500 rpm for 15 min
- Re-suspend the cell pellets in 4mL of fresh LB.
- Inoculate the expression culture media by dividing the resuspended cells among the Fernbach flasks.
- Incubate the expression cultures at 37C and 160rpm until the absorbance of the media at 600 nm = 0.6.
- Add 1 mL of 0.4 M IPTG to each flask in order to induce protein expression
- Continue shaking for 3-4 hr
- Harvest the cells by centrifuging at 4500 rpm for 15 min at 4°C with a SH-3000(BK) rotor.
- 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
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