User:Nader A. Khamis/Notebook/Experimental Biological Chemistry AU Fall 2011/2011/10/05

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  • Use Ru(bpy)(phen) in acetonitrile to determine the sensitivity of the Fluorometer.
  • Obtain fluorescence spectrum of Maltose Binding Protein (MBP).
  • Purify DNA from experiment performed on September 27th,2011


Six 3ml Ru(bpy)(phen) Standards were prepared:

0 uM - Only 3 mL of Acetanitrile

20 uM - 0.126 mL Ru(bpy) and 2.874 mL Acetanitrile

40 uM - 0.252 mL Ru(bpy) and 2.748 mL Acetanitrile

60 uM - 0.378 mL Ru(bpy) and 2.622 mL Acetanitrile

80 uM - 0.500 mL Ru(bpy) and 2.496 mL Acetanitrile

100 uM - 0.630 mL Ru(bpy) and 2.37 mL Acetanitrile

Solutions A - E were prepared to determine fluorescence of Maltose Binding Protein (MBP).

A - 1397 uL MBP and 1603 uL Tris Buffer pH 7.55

B - 300 uL Solution A and 2700 uL Tris

C - 300 uL Solution B and 2700 uL Tris

D - 300 uL Solution C and 2700 uL Tris

E - 300 uL Solution C and 2700 uL Tris

The spectra of the five solutions were then measured from 310 - 500 nm with excitation at 290 nm

  • The procedure for DNA purification was from "Wizard® Plus SV Minipreps DNA Purification System":



  • According to the graph below,in which Flourescence intensity is plotted as a function of wavelength, the 60uM Ru(bpy)(phen) standard exhibited the highest intensity.

Experimental Biological Chemistry - Picture 1 - October 2011.jpg

  • The graph below is a plot of integrated intensity as a function of Ru(bpy)(phen)concentration.
  • X and Y values for the graph below are:

0 uM - 11.9577785

20 uM - 1742.89187

40 uM - 2239.60778

60 uM - 2462.69942

80 uM - 2474.32617

100 uM - 2391.34271

Experimental Biological Chemistry - October 5th 2011....jpg

  • The following graph shows the intensities of the five MBP solutions prepared plotted against the wavelengths.
  • MBP Solutions: A, B, C, D, and E

Experimental Biological Chemistry - Picture 3 - October 2011.jpg

  • The graph below is a plot of Absorbance spectra of MBP Solutions A, B, C, D, and E as a function of wavelength.

Experimental Biological Chemistry - Solutions November 2011.jpg

  • Concentrations of the five MBP solutions were calculated using the molar absorptivity of MPB which has been determined on September 14th 2011 and the absorbance spectra of MBP at 280nm:

Solution A= 1.09306*10-5 M

Solution B= 1.08887*10-6 M

Solution C= 1.67518*10-7 M

Solution D= 4.18796*10-8 M

Solution E= 2.09398*10-8 M


  • The curves in Solution A and B become flat because flourometer cannot measure the intensity of MBP in solution A and B. The concentrations of Solution A and B are too high for the flourometer to read.
  • To avoid negative concentration values, the base lines for solutions D and E were slightly altered.

Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized. .