User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/25: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
(10 intermediate revisions by the same user not shown)
Line 8: Line 8:
==July 25, 2013==
==July 25, 2013==
===Procedure Update: MHA Silica-Coating===
===Procedure Update: MHA Silica-Coating===
According to [http://link.springer.com/content/pdf/10.1007%2Fs11051-012-0758-z.pdf Aggregation and adhesion of gold nanoparticles in phosphate buffer saline], PBS and prolonged sonication actually cause aggregation in gold nanoparticle solutions: "results showed an optimized sonication time of 10 min to reach equilibrium for plain gold nanoparticles in water."  Instead of using PBS to dilute all components, filtered MilliQ water will be used to dilute nanoparticles as well as MHA, as the organic solvent in solution prior to the actual silica-coating may have also been affecting observed the gold nanoparticle aggregation.
According to [http://link.springer.com/content/pdf/10.1007%2Fs11051-012-0758-z.pdf Aggregation and adhesion of gold nanoparticles in phosphate buffer saline], PBS and prolonged sonication actually cause aggregation in gold nanoparticle solutions: "results showed an optimized sonication time of 10 min to reach equilibrium for plain gold nanoparticles in water."  Instead of using PBS to dilute all components, filtered MilliQ water will be used to dilute nanoparticles as well as MHA, as the organic solvent in solution prior to the actual silica-coating may have also been affecting observed the gold nanoparticle aggregation.  Filtered MilliQ water will be brought up to room temperature as we've noticed cold solutions (whether water or PBS) have seemed to induce aggregation as well.
*Originally detailed on [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/05/24 05/24/2013]
*Originally detailed on [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/05/24 05/24/2013]
*Last updated on [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/07/12#Procedure_Update:_Silica-coating_AuNPs_by_MHA 07/12/2013]
*Last updated on [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/07/12#Procedure_Update:_Silica-coating_AuNPs_by_MHA 07/12/2013]
*Day One:
*Day One:
**Attach MHA to AuNPs: add 5mL of 10mM MHA to 5mL of AuNPs, stir overnight
**Attach MHA to AuNPs: add 5mL of 10mM MHA to 5mL of AuNPs
***To prepare 10mM MHA solution:  
***To prepare 5mL of 10mM MHA solution:  
***(5 mL)×(L/1000mL)×(10mmoles/L)×(1mol/1000mmoles)×(288.49g/mol)=0.0144g MHA in 5mL filtered MilliQ water
***(5 mL)×(L/1000mL)×(10mmoles/L)×(1mol/1000mmoles)×(288.49g/mol)=0.0144g MHA in 5mL filtered MilliQ water
***To prepare 5mL of 9.4nM AuNPs:
***To prepare 5mL of 9.4nM AuNPs:
***(29.67nM)V=(9.4nM)(5mL)
***(23.5nM)V=(9.4nM)(5mL)
***V=1.584mL AuNPs in 3.416mL filtered MilliQ water
***V=2mL AuNPs in 3mL filtered MilliQ water
***Combine MHA and AuNP solutions
***Combine MHA and AuNP solutions
**Stir overnight
**Note: heat to 130C for approximately five minutes if MHA does not dissolve (when first added and before silica-coating reaction; do not heat overnight)
*Day Two:
*Day Two:
**Prepare the following solutions:
**Prepare the following solutions:
#1mL 9.4nM, 10nm AuNP&MHA/20μL TES/0.125mL NH4OH/5mL propanol
#1mL 9.4nM, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/5μL TES/0.125mL NH<sub>4</sub>OH/5mL isopropanol
#1mL 9.4nM, 10nm AuNP&MHA/15μL TES/0.125mL NH4OH/5mL propanol
#1mL 9.4nM, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/4μL TES/0.125mL NH<sub>4</sub>OH/5mL isopropanol
#1mL 9.4nM, 10nm AuNP&MHA/10μL TES/0.125mL NH4OH/5mL propanol
#1mL 9.4nM, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/3μL TES/0.125mL NH<sub>4</sub>OH/5mL isopropanol
#1mL 9.4nM, 10nm AuNP&MHA/5μL TES/0.125mL NH4OH/5mL propanol
#1mL 9.4nM, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/2μL TES/0.125mL NH<sub>4</sub>OH/5mL isopropanol
#1mL 9.4nM, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/1μL TES/0.125mL NH<sub>4</sub>OH/5mL isopropanol
#*Stir solutions for one hour to allow reaction to proceed
#*Stir solutions for one hour to allow reaction to proceed
#*Centrifuge solutions at 4000 rpm for one hour
#*Centrifuge solutions at 4000 rpm for one hour
#*Re-suspend particles in 140uL PBS (per solution)
#*Re-suspend particles in 140uL PBS (per solution)
*Note: two samples of MHA/AuNPs were made: MHA dissolved in water & heated, MHA in isopropanol & not heated
===Fluorescence Data: 4 and 8 Repeat DNA with ThT===
Fluorescence measurements taken and calibration curve constructed in order to show the increased fluorescence intensity with increases number of G-quadruplexes formed.<br.>
[[Image:2013_0725_ThT.PNG]]<br.>
[[Image:2013_0725_4DNA.PNG]]<br.>
[[Image:2013_0725_8DNA.PNG]]<br.>
<br.>
[[Image:2013_0725_4_and_8_DNA_w_ThT_calibration.PNG]]<br.>
<br.>


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 12:38, 25 July 2013

Procedure Update, MHA Day One <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

July 25, 2013

Procedure Update: MHA Silica-Coating

According to Aggregation and adhesion of gold nanoparticles in phosphate buffer saline, PBS and prolonged sonication actually cause aggregation in gold nanoparticle solutions: "results showed an optimized sonication time of 10 min to reach equilibrium for plain gold nanoparticles in water." Instead of using PBS to dilute all components, filtered MilliQ water will be used to dilute nanoparticles as well as MHA, as the organic solvent in solution prior to the actual silica-coating may have also been affecting observed the gold nanoparticle aggregation. Filtered MilliQ water will be brought up to room temperature as we've noticed cold solutions (whether water or PBS) have seemed to induce aggregation as well.

  • Originally detailed on 05/24/2013
  • Last updated on 07/12/2013
  • Day One:
    • Attach MHA to AuNPs: add 5mL of 10mM MHA to 5mL of AuNPs
      • To prepare 5mL of 10mM MHA solution:
      • (5 mL)×(L/1000mL)×(10mmoles/L)×(1mol/1000mmoles)×(288.49g/mol)=0.0144g MHA in 5mL filtered MilliQ water
      • To prepare 5mL of 9.4nM AuNPs:
      • (23.5nM)V=(9.4nM)(5mL)
      • V=2mL AuNPs in 3mL filtered MilliQ water
      • Combine MHA and AuNP solutions
    • Stir overnight
    • Note: heat to 130C for approximately five minutes if MHA does not dissolve (when first added and before silica-coating reaction; do not heat overnight)
  • Day Two:
    • Prepare the following solutions:
  1. 1mL 9.4nM, 9.65nm AuNP&MHA, MQ H2O/5μL TES/0.125mL NH4OH/5mL isopropanol
  2. 1mL 9.4nM, 9.65nm AuNP&MHA, MQ H2O/4μL TES/0.125mL NH4OH/5mL isopropanol
  3. 1mL 9.4nM, 9.65nm AuNP&MHA, MQ H2O/3μL TES/0.125mL NH4OH/5mL isopropanol
  4. 1mL 9.4nM, 9.65nm AuNP&MHA, MQ H2O/2μL TES/0.125mL NH4OH/5mL isopropanol
  5. 1mL 9.4nM, 9.65nm AuNP&MHA, MQ H2O/1μL TES/0.125mL NH4OH/5mL isopropanol
    • Stir solutions for one hour to allow reaction to proceed
    • Centrifuge solutions at 4000 rpm for one hour
    • Re-suspend particles in 140uL PBS (per solution)
  • Note: two samples of MHA/AuNPs were made: MHA dissolved in water & heated, MHA in isopropanol & not heated

Fluorescence Data: 4 and 8 Repeat DNA with ThT

Fluorescence measurements taken and calibration curve constructed in order to show the increased fluorescence intensity with increases number of G-quadruplexes formed.<br.> <br.> <br.> <br.> <br.> <br.> <br.>


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/07/25&oldid=712316"