User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/21

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Meeting, Absorbance Data</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Entry title==
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==June 21, 2013==
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* Insert content here...
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===Notes from Meeting===
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*Check pH of sodium citrate buffer (color change?)
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**Read literature for salt effects on AuNPs
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*Make lower concentration dilutions of DNA/ThT solutions by serial dilutions
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**Plot the ratio rather than the DNA concentration
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*Use the same wavelength for the calibration curve; take zero point (ThT) out of the calibration curve
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*Tuesday: run MB samples on the FCS with Dr Miller
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*Dilute 10x AuNPs@SiO<sub>2</sub>
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**Read literature for alternative detection methods
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*Endnote
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===Absorbance Data===
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[[Image:Abs_data_2013_0621_AuNPs@SiO2_MHA.png]]<br.>
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<br.>
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[[Image:Abs_data_2013_0621_AuNPs@SiO2_20uL.png]]<br.>
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<br.>
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[[Image:Abs_data_2013_0621_AuNPs@SiO2_40uL.png]]<br.>
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<br.>
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====Notes====
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Absorbance spectra, especially those from the silica-coated AuNPs made with MHA and 5uL TES, are consistent with those from the literature (according to [http://pubs.acs.org/doi/full/10.1021/la9601871 Synthesis of Nanosized Gold−Silica Core−Shell Particles]).  The silica-coated AuNPs show a peak at approximately 525nm.<br.>
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<br.>
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Since the AuNPs@SiO<sub>2</sub> synthesized with MHA and 5uL TES produced the best spectra, I think I will move onto the DNA attachment procedure using those.  Additionally, I'm interested in varying the volume of TES added to see if higher or lower volumes will produce thicker or thinner silica-coatings, respectively (keeping the MHA constant and trying 10uL, 15uL, 20uL).  I might also look at the volumes of TES used without MHA: since the higher volume of TES (40uL) did not seem to produce the expected spectra and the lower volume (20uL) did, I'd be interested to see the results of using 30uL, 15uL, 10uL, and 5uL of TES would be.<br.>
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<br.>
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A final important note- PBS was used for ALL dilutions in this procedure, and sample were sonicated after dilution before being measured.  The salt content of the PBS prevents the nanoparticles from aggregating.
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===DNA/ThT Solutions===
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Solutions of 100nM, 200nM and 700nM DNA with 2.5uM ThT were prepared and their fluorescence measured to make a more complete calibration curve (see [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/06/20 06/20/2013]). Fluorescence for the 200nM sample observed as expected, but the 100nM was much lower than expected and the 700nM much higher than expected.  This inconsistency may be due to incorrect calibration of pipetman. Samples will be made again and run next week.
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Current revision

Meeting, Absorbance Data Main project page
Previous entry      Next entry

June 21, 2013

Notes from Meeting

  • Check pH of sodium citrate buffer (color change?)
    • Read literature for salt effects on AuNPs
  • Make lower concentration dilutions of DNA/ThT solutions by serial dilutions
    • Plot the ratio rather than the DNA concentration
  • Use the same wavelength for the calibration curve; take zero point (ThT) out of the calibration curve
  • Tuesday: run MB samples on the FCS with Dr Miller
  • Dilute 10x AuNPs@SiO2
    • Read literature for alternative detection methods
  • Endnote

Absorbance Data

Image:Abs_data_2013_0621_AuNPs@SiO2_MHA.png

Image:Abs_data_2013_0621_AuNPs@SiO2_20uL.png

Image:Abs_data_2013_0621_AuNPs@SiO2_40uL.png

Notes

Absorbance spectra, especially those from the silica-coated AuNPs made with MHA and 5uL TES, are consistent with those from the literature (according to Synthesis of Nanosized Gold−Silica Core−Shell Particles). The silica-coated AuNPs show a peak at approximately 525nm.

Since the AuNPs@SiO2 synthesized with MHA and 5uL TES produced the best spectra, I think I will move onto the DNA attachment procedure using those. Additionally, I'm interested in varying the volume of TES added to see if higher or lower volumes will produce thicker or thinner silica-coatings, respectively (keeping the MHA constant and trying 10uL, 15uL, 20uL). I might also look at the volumes of TES used without MHA: since the higher volume of TES (40uL) did not seem to produce the expected spectra and the lower volume (20uL) did, I'd be interested to see the results of using 30uL, 15uL, 10uL, and 5uL of TES would be.

A final important note- PBS was used for ALL dilutions in this procedure, and sample were sonicated after dilution before being measured. The salt content of the PBS prevents the nanoparticles from aggregating.

DNA/ThT Solutions

Solutions of 100nM, 200nM and 700nM DNA with 2.5uM ThT were prepared and their fluorescence measured to make a more complete calibration curve (see 06/20/2013). Fluorescence for the 200nM sample observed as expected, but the 100nM was much lower than expected and the 700nM much higher than expected. This inconsistency may be due to incorrect calibration of pipetman. Samples will be made again and run next week.



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