User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/10/05

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Objective

The purpose of today's experiment was to use standard concentrations Ru(bpy)(phen) to determine the sensitivity of a fluorimeter. This was then used to determine the concentration of a stock solution of maltose binding protein (MBP).

The cells cultured on 09/27/11 also underwent DNA purification as well.

Procedure

MBP Concentration Determination:

  • 5 varying concentrations of MBP were prepared using TRIS as the solvent. The 5 solutions each were composed of:
Solution Conc.jpg

So that each following stock solution had a dilution of MBP by a factor of 10.

  • These solutions were then analyzed using both UV-Vis and a fluorimeter. The intensities were taken from 310nm-500nm using an excitation value of 290nm.
  • The data from the UV-Vis was analyzed and a graph containing absorbance vs. concentration was created.
  • The absorbance of each of the solutions at 280 nm was used to calculate the concentration of MBP. This was done using the epsilon value found from [1], which was determined to be 334579.807 (1/M*cm) at 280 nm. The corresponding peak from these calculations was:
EpsilonZoom22.jpg
  • A graph of intensity/concentration of MBP vs. wavelength for each solution was created and used for analysis.

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

Results

The final concentration of MBP in each of the stock solutions was determined to be:

Final Concentrations.jpg

This was determined using the values from the UV Vis analysis. The resulting absorbance vs. wavelength graph yielded:

UVVISMBP.jpg

Each solution (A-E) was graphed as Intensity/Concentration vs. Wavelength.

IC A.jpg


IC B.jpg

The resulting graphs of both Solution A and Solution B show that the concentration of the MBP was too high to be detected on the fluorimeter.

IC C.jpg
IC D.jpg
IC E.jpg

Each of these graphs had a maximum that was determined to be:

Peaky.jpg
  • The values for Stock Solutions A and B should not be factored in as their peaks were unchartable.
Groupdata3.jpg

This graph shows the collective data of every group participating in this experiment. This graph will be updated as more groups record their information. Group 3, which is denoted by asterisks, is the group that this notebook's information is from.

Notes

  • A baseline change was preformed for the data results from Stock E. An value of 0.004 was added to each data point to prevent a negative concentration.