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

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Objective

  • 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

Description

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":

[1]

Data

  • 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.

Image: 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


Image: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

Image: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.

Image: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

Notes

  • 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.

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