User:Elaine Marie Robbins/Notebook/Capstone/2012/06/20

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Methods

MB concentration effects

  1. Mix 3 μL AuNP/TS primers, 1 μL dNTP, 2.5 μL PCR buffer, and 250 μL diluted cell extract containing 100,000 cells.
  2. Incubate at 30°C for 30 min.
  3. Add MB to each solution:
    • 12.2 μL 100x diluted MB for MB/primers = 1
    • 1.22 μL concentrated MB for MB/primers = 10
    • 6.11 μL concentrated MB for MB/primers = 50
  4. Incubate at 37°C for 60 min.

Primers vs AuNP, incubation vs no incubation

  1. Make 4 solutions, each with 100,000 cells:
    • Citrate AuNP/TS primer, incubated
      1. Mix 3 μL AuNP/TS primers, 1 μL dNTP, 2.5μL PCR buffer, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at 30°C for 30 min.
      3. Add 1.22 μL MB.
      4. Incubate at 37°C for 60 min.
    • Citrate AuNP/TS primer, kept at room temperature
      1. Mix 3 μL AuNP/TS primers, 1 μL dNTP, 2.5μL PCR buffer, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at room temperature for 30 min.
      3. Add 1.22 μL MB.
      4. Incubate at 37°C for 60 min.
    • Primers only, incubated
      1. Mix 6.5 μL 100x diluted primers, 1 μL dNTP, 2.5μL PCR buffer, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at 30°C for 30 min.
      3. Add 1.22 μL MB.
      4. Incubate at 37°C for 60 min.
    • Primers only, kept at room temperature
      1. Mix 6.5 μL 100x diluted primers, 1 μL dNTP, 2.5μL PCR buffer, and 250 μL diluted cell extract containing 100,000 cells.
      2. Incubate at room temperature for 30 min.
      3. Add 1.22 μL MB.
      4. Incubate at 37°C for 60 min.
  2. Take fluorescence data for each solution.

Data

Image:12-06-20 fluorescence MB concentration effects.png
Image:12-06-20 fluorescence of AuNP vs primers, incubation vs room temp.png


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