User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/10/05

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Objective

  • Determine the sensitivity of the Fluorometer using Ru(bpy)(phen) in acetonitrile.
  • Determine Fluorescence of Maltose Binding Protein (MBP)
  • Purify DNA from experiment performed on 9/27/2011

Description

  • Ru(bpy)(phen) in Acetonitrile standards were made to determine sensitivity of instrument.
  • Solutions A - E were prepared to determine fluorescence of Maltose Binding Protein (MBP).
  • Ru(bpy)(phen) Standards (3 mL Total):

0 μM - 3 mL Acetanitrile

20 μM - .126 mL Ru(bpy) and 2.874 mL Acetanitrile

40 μM - .252 mL Ru(bpy) and 2.748 mL Acetanitrile

60 μM - .378 mL Ru(bpy) and 2.622 mL Acetanitrile

80 μM - .500 mL Ru(bpy) and 2.496 mL Acetanitrile

100 μM - .630 mL Ru(bpy) and 2.37 mL Acetanitrile

  • Spectra for the standards were measured at 300 - 700 nm with excitation at 450 nm
  • MBP Solutions (3 mL Total):

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

B - 300 μL Solution A and 2700 μL Tris

C - 300 μL Solution B and 2700 μL Tris

D - 300 μL Solution C and 2700 μL Tris

E - 300 μL Solution C and 2700 μL Tris

  • Spectra for the solutions were measured at 310 - 500 nm with excitation at 290 nm
  • DNA Purification procedure was from "Wizard® Plus SV Minipreps DNA Purification System":

[1]

Data

  • Using excel, the curves were integrated
  • Integrated Intensities:


0 μM - 11.9577785

20 μM - 1742.89187

40 μM - 2239.60778

60 μM - 2462.69942

80 μM - 2474.32617

100 μM - 2391.34271


  • Fluorescence intensity values were plotted against wavelength. Near 60 μM, Ru(bpy)(phen) emission is at it's maximum.

OC-10-5-1.jpg

  • Plot of integrated intensity against concentration of Ru(bpy)(phen).

OC-10-5-2.jpg


  • Fluorescence spectra of MBP Solutions A, B, C, D, and E:


OC-10-5-3.jpg

  • Absorbance spectra of MBP Solutions A, B, C, D, and E.


OC-10-5-4.jpg

  • Using the absorbance spectra of the MBP solutions at 280 nm, along with the molar absorptivity of MBP calculated on 9/14/2011, the concentration of MBP in each solution was calculated.


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

  • Intensity/Concentration of each MBP solution was plotted against the wavelength.


OC-10-5-5.jpg


OC-10-5-6.jpg


OC-10-5-7.jpg


OC-10-5-8.jpg


OC-10-5-9.jpg

Notes

  • Solutions A and B have concentrations of MBP that are too high for the flourimeter to read which is why their curves are flat.
  • The baselines for solutions D and E were changed slightly to avoid having negative concentrations.