User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/09/27

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Objective

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.

Description

Digestion of Wild-type DNA and Transformation

  1. Add 1μL of DpnI to Group 2's PCR product from last week. Place this at 37°C for 1 hour.
  2. Remove the PCR product and place on ice.
  3. Take a sterile eppendorf tube and place it on ice.
  4. Mix 40μL of NovaBlue Competent E.coli with 5μL of the PCR product in the cold, sterile tube.
  5. Incubate this mixture for 30 minutes on ice.
  6. Transfer the tube to a heat block at 42°C for 30 seconds, and then transfer the tube back to ice for 5 minutes.
  7. Add 250μL of SOC media to the cells/PCR product.
  8. Shake the mixture at 250rpm at 37°C for one hour.
  9. Spread 100μL of the mixture on an LB plate with 100μg/mL.
  10. Incubate the plate overnight (inverted) at 37°C.

Biominalization of Gold Nanoparticles

  1. Mix 8mL 50mM Acetate buffer, pH 3.6, 1mL 15.4μM BSA, and 580μL of 4.3mM Chloroauric acid.
  2. 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.
  3. Put the rest of the solution in the oven set to 80°C for about 3.5 hours.

Data

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:

Notes

  • The final concentration of BSA is 1.61μM.
    • Calculation:(1mL/9.58mL)*15.4μM
  • 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.



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