User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/10/05

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Objective

To continue the experiment in determining the sensitivity of the fluorimeter and to purify DNA made by cells that were cultured on 09/27/11.

Description

1. Fluorimeter Sensitivity II

  • Integrate the intensity values and graph them against concentration.
  1. 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
  2. Take fluorescence spectra of each dilution.


2. DNA Purification

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

http://www.promega.com/~/media/Files/Resources/ProtCards/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Quick%20Protocol.ashx

Fluorimeter Sensitivity II

Data

This graph shows the integral of fluorescence intensity vs. the concentration of Ru(bpy)(phen). The graph begins to level off, indicating that the Ru(bpy)(phen) is emitting too much light for the fluorimeter.

Image:Fluorescence_v._concentration.png


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

Notes


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