Biomineralization of gold nanoparticles while keeping the solution at 80°C the entire time. Also, Group 2's PCR product will have the wild-type DNA digested by DpnI, and then the mutated DNA will be transformed into E. coli.
Digestion of Wild-type DNA and Transformation
- Add 1μL of DpnI to Group 2's PCR product from last week. Place this at 37°C for 1 hour.
- Remove the PCR product and place on ice.
- Take a sterile eppendorf tube and place it on ice.
- Mix 40μL of NovaBlue Competent E.coli with 5μL of the PCR product in the cold, sterile tube.
- Incubate this mixture for 30 minutes on ice.
- Transfer the tube to a heat block at 42°C for 30 seconds, and then transfer the tube back to ice for 5 minutes.
- Add 250μL of SOC media to the cells/PCR product.
- Shake the mixture at 250rpm at 37°C for one hour.
- Spread 100μL of the mixture on an LB plate with 100μg/mL.
- Incubate the plate overnight (inverted) at 37°C.
Biominalization of Gold Nanoparticles
- Mix 8mL 50mM Acetate buffer, pH 3.6, 1mL 15.4μM BSA, and 580μL of 4.3mM Chloroauric acid.
- Take 1mL of this solution and transfer it to a quartz cuvette, and place the cuvette in the Shimadzu UV-2550 spectrophotometer at 70°C.
- Take a spectrum from 200nm-800nm of this every half hour.
- Put the rest of the solution in the oven set to 80°C for about 3.5 hours.
A spectrum of the BSA/gold mixture in the quartz cuvette was taken every half hour, and the temperature of the mixture stayed at 70°C. The absorbance was corrected with a buffer spectrum that was taken on 9/7/11, of the same buffer used. The spectra are graphed:
Because the nanoparticle formation is seen at 550nm, the spectra were zoomed in to better see the increase in absorbance at this wavelength:
The absorbance at 550nm is graphed against time, and it is seen that the absorbance is clearly increasing at 550nm in the mixture:
- The final concentration of BSA is 1.61μM.
- The final concentration of Chloroauric acid is 0.26mM.
- Calculation: (.58mL/9.58mL)*4.3mM
- Purple fibers formed again in both the test tube and the quartz cuvette. It also appeared as though the liquid in the quartz cuvette had turned a light purple color.