User:Elaine Marie Robbins/Notebook/CHEM-496/2011/10/05

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Fluorimeter Sensitivity II, DNA Purification Main project page
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Fluorimeter Sensitivity II

To continue the experiment on 10/04/11 to determine the sensitivity of a fluorimeter using standard concentrations of Ru(bpy)(phen) and determine the fluorescence of MBP as a function of concentration.

DNA Purification

To purify the DNA made by cells cultured on 09/27/11.


Fluorimeter Sensitivity II

  1. Integrate the intensity values found on 10/05/11 and graph them against concentration.
  2. Make dilutions of MBP in 50 mM Tris buffer (7.55 pH).
    • Blank: 50 mM Tris (7.55 pH)
    • A: 1397 μM MBP stock + 1603 μM Tris
    • B: 300 μM A + 2700 μM Tris
    • C: 300 μM B + 2700 μM Tris
    • D: 300 μM C + 2700 μM Tris
    • E: 300 μM D + 2700 μM Tris
  3. Take fluorescence spectra of each dilution.

DNA Purification

The DNA purification protocol was adapted from the Wizard Plus SV Minpreps DNA Purification System vacuum protocol found here:


This graph shows the integral of fluorescence intensity vs. the concentration of Ru(bpy)(phen). The graph begins to level off at 60 μM, indicating that the Ru(bpy)(phen) is emitting too much light for the fluorimeter at around that point. This is consistent with the findings on 10/04/11

UV vis data was taken for the MBP samples, and a graph of molar absorptivity vs. wavelength was made. Using the data obtained from the UV vis, the concentrations of the MBP dilutions were calculated.


Calculated concentrations of MBP dilutions:

  • A 1.6700×10-6 M
  • B 3.0895×10-7 M
  • C 2.3911×10-8 M
  • D 5.6788×10-8 M
  • E 8.9665×10-9 M

Graphs of the corrected intensity of fluorescence vs. wavelength for each concentration of MBP were created.


Integrated intensity of Ru(bpy)(phen):

  • 20 μM: 1432.805647
  • 40 μM: 2001.315233
  • 60 μM: 2237.400074
  • 80 μM: 2294.581483
  • 100 μM: 2286.935912

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