User:Elaine Marie Robbins/Notebook/Capstone/2012/06/19

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Owwnotebook icon.png Concentration of AuNP <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Methods

  1. Determine size and concentration of citrate AuNP using UV/Vis.
  2. Make AuNP/TS primer complex with approx. 50 primers per AuNP by adding 40.5 μL thiol primers to 5 mL AuNP.
  3. Make 4 solutions, each with 100,000 cells:
    • Citrate AuNP/TS primer, incubated
      1. Mix 3 μL AuNP/TS primers, 3.5 μL 10 mM tris buffer (pH 7.5), 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at 30°C for 30 min.
      3. Add 12.2 μL 100x diluted MB.
      4. Incubate at 65°C for 60 min.
    • Citrate AuNP/TS primer, kept at room temperature
      1. Mix 3 μL AuNP/TS primers, 3.5 μL 10 mM tris buffer (pH 7.5), 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at room temperature for 30 min.
      3. Add 12.2 μL 100x diluted MB.
      4. Incubate at 65°C for 60 min.
    • Primers only, incubated
      1. Mix 6.5 μL 100x diluted primers, 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at 30°C for 30 min.
      3. Add 12.2 μL 100x diluted MB.
      4. Incubate at 65°C for 60 min.
    • Primers only, kept at room temperature
      1. Mix 6.5 μL 100x diluted primers, 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at room temperature for 30 min.
      3. Add 12.2 μL 100x diluted MB.
      4. Incubate at 65°C for 60 min.
  4. Take fluorescence data for each solution.
  5. Repeat, only incubate at 37°C instead of 65°C, don't add water, and add 2.5 μL of PCR buffer to each solution except one.

Data

12-06-19 uv-vis of citrate aunp.png


Max absorbance 2.954
Wavelength (nm) 523
Absorbance at 450 nm 1.861
Aspr/A450 1.587318646
Approx AuNP diameter (nm) 13
AuNP surface area (nm2) 530.9135
ε450 nm (1/(M*cm)) 1.39*108
AuNP concentration (M) 1.34*10-8


  • Fluorescence of samples incubated at 65°C:
12-06-19 fluorescence incubated at 65C.png
  • Fluorescence of samples incubated at 37°C:
12-06-19 fluorescence incubated at 37C.png