User:Elaine Marie Robbins/Notebook/CHEM-496/2011/10/05: Difference between revisions
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<b>Fluorimeter Sensitivity II</b> | <b>Fluorimeter Sensitivity II</b> | ||
To continue the experiment on [http://openwetware.org/wiki/User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/04 10/04/11] to determine the sensitivity of a fluorimeter using standard concentrations of Ru(bpy)(phen). | To continue the experiment on [http://openwetware.org/wiki/User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/04 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. | ||
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==Description== | ==Description== | ||
<b>Fluorimeter Sensitivity II</b> | |||
The DNA purification protocol was adapted from the Wizard Plus SV Minpreps DNA Purification System found here: | # Integrate the intensity values found on [http://openwetware.org/wiki/User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/05 10/05/11] and graph them against concentration. | ||
# 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 | |||
# Take fluorescence spectra of each dilution. | |||
<b>DNA Purification</b> | |||
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 | http://www.promega.com/~/media/Files/Resources/ProtCards/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Quick%20Protocol.ashx | ||
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==Data== | ==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 [http://openwetware.org/wiki/User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/04 10/04/11] | |||
<center>[[Image:Integral of intensity vs concentration.jpg|600px]]</center> | |||
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. | |||
<center>[[Molar_absorptivity_vs_wavelength_10-5-11.jpg|600px]]</center> | |||
Calculated concentrations of MBP dilutions: | |||
* A 1.6700×10<sup>-6</sup> M | |||
* B 3.0895×10<sup>-7</sup> M | |||
* C 2.3911×10<sup>-8</sup> M | |||
* D 5.6788×10<sup>-8</sup> M | |||
* E 8.9665×10<sup>-9</sup> M | |||
Graphs of the corrected intensity of fluorescence vs. wavelength for each concentration of MBP were created. | |||
<center>[[Image:Intensity over concentration vs wavelength a 10-5-11.jpg|600px]]</center> | |||
<center>[[Image:Intensity over concentration vs wavelength b 10-5-11.jpg|600px]]</center> | |||
<center>[[Image:Intensity over concentration vs wavelength c 10-5-11.jpg|600px]]</center> | |||
<center>[[Image:Intensity over concentration vs wavelength d 10-5-11.jpg|600px]]</center> | |||
<center>[[Image: | <center>[[Image:Intensity over concentration vs wavelength e 10-5-11.jpg|600px]]</center> | ||
==Notes== | ==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 | |||
Revision as of 13:42, 30 November 2011
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ObjectiveFluorimeter 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.
To purify the DNA made by cells cultured on 09/27/11. DescriptionFluorimeter Sensitivity II
DNA Purification The DNA purification protocol was adapted from the Wizard Plus SV Minpreps DNA Purification System vacuum protocol found here: DataThis 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
NotesIntegrated intensity of Ru(bpy)(phen):
|