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

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(Absorbance Data: 4 and 8 Repeat DNA with ThT)
Current revision (15:38, 25 July 2013) (view source)
(July 25, 2013)
 
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*Day Two:
*Day Two:
**Prepare the following solutions:
**Prepare the following solutions:
-
#1mL 9.4nM, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/5μL TES/0.125mL NH4OH/5mL isopropanol
+
#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, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/4μL TES/0.125mL NH4OH/5mL isopropanol
+
#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, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/3μL TES/0.125mL NH4OH/5mL isopropanol
+
#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, 9.65nm AuNP&MHA, MQ H<sub>2</sub>O/2μL TES/0.125mL NH4OH/5mL isopropanol
+
#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 NH4OH/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)
-
===Absorbance Data: 4 and 8 Repeat DNA with ThT===
+
*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_ThT.PNG]]<br.>
[[Image:2013_0725_4DNA.PNG]]<br.>
[[Image:2013_0725_4DNA.PNG]]<br.>

Current revision

Procedure Update, MHA Day One Main project page
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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.
Image:2013_0725_ThT.PNG
Image:2013_0725_4DNA.PNG
Image:2013_0725_8DNA.PNG

Image:2013_0725_4_and_8_DNA_w_ThT_calibration.PNG


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