User:Elaine Marie Robbins/Notebook/CHEM-496/2011/10/05

From OpenWetWare

< User:Elaine Marie Robbins | Notebook | CHEM-496 | 2011 | 10
Revision as of 15:42, 30 November 2011 by Elaine Marie Robbins (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search
Fluorimeter Sensitivity II, DNA Purification Main project page
Previous entry      Next entry


Objective

Fluorimeter Sensitivity II

To continue the experiment on 10/04/11 to determine the sensitivity of a fluorimeter using standard concentrations of Ru(bpy)(phen) and determine the fluorescence of MBP as a function of concentration.


DNA Purification

To purify the DNA made by cells cultured on 09/27/11.

Description

Fluorimeter Sensitivity II

  1. Integrate the intensity values found on 10/05/11 and graph them against concentration.
  2. 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
  3. Take fluorescence spectra of each dilution.

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

Data

This graph shows the integral of fluorescence intensity vs. the concentration of Ru(bpy)(phen). The graph begins to level off at 60 μM, indicating that the Ru(bpy)(phen) is emitting too much light for the fluorimeter at around that point. This is consistent with the findings on 10/04/11



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.


600px


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.

Notes

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




Personal tools