User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/12

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(Procedure Update: Silica-coating AuNPs by MHA)
Current revision (08:48, 22 July 2013) (view source)
(Procedure Update: Silica-coating AuNPs by MHA)
 
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**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, stir overnight
***To prepare 10mM MHA solution:  
***To prepare 10mM MHA solution:  
-
***(5 mL)(L/1000mL)(10mmoles/L)(1mol/1000mmoles)(288.49g/mol)=0.0144g MHA in 5mL
+
***(5 mL)×(L/1000mL)×(10mmoles/L)×(1mol/1000mmoles)×(288.49g/mol)=0.0144g MHA in 5mL 2-propanol
 +
***To prepare 5mL of 9.4nM AuNPs:
 +
***(29.67nM)V=(9.4nM)(5mL)
 +
***V=1.584mL AuNPs in 3.416mL PBS buffer
 +
***Combine MHA and AuNP solutions
*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, 10nm AuNP&MHA/15μL TES/0.125mL NH4OH/5mL propanol
 +
#1mL 9.4nM, 10nm AuNP&MHA/10μL TES/0.125mL NH4OH/5mL propanol
#1mL 9.4nM, 10nm AuNP&MHA/5μL TES/0.125mL NH4OH/5mL propanol
#1mL 9.4nM, 10nm AuNP&MHA/5μL TES/0.125mL NH4OH/5mL propanol
-
#1mL 9.4nM, 10nm AuNP&MHA/4μL TES/0.125mL NH4OH/5mL propanol
 
-
#1mL 9.4nM, 10nm AuNP&MHA/3μL TES/0.125mL NH4OH/5mL propanol
 
-
#1mL 9.4nM, 10nm AuNP&MHA/2μL TES/0.125mL NH4OH/5mL propanol
 
-
#1mL 9.4nM, 10nm AuNP&MHA/1μL TES/0.125mL NH4OH/5mL propanol
 
#*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 PBS to a final concentration of 5nM?
+
#*Re-suspend particles in 140uL PBS (per solution)

Current revision

Meeting, DNA/AuNP Attachment Main project page
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July 12, 2013

Notes from Meeting

  • Adjust range and use SymphoTime to better fit FCS graphs in Igor
  • Use error bars on calibration graphs (for Igor <N> values)
  • Focus on attaching AuNPs to DNA & hybridizing MB to run on FCS
  • Use a range of volumes of TES in MHA silica-coating procedure to change silica-coating thickness
    • Take absorbance data to see differences between solution, then use TEM imaging to verify presence of silica-coating
  • Presentation at the end of 10 weeks

DNA and AuNP Attachment Procedure

Calculations

  • Done by Allison Alix.
  • Extraction Procedure:
    • Combine 250uL 4.45uM DNA (diluted in PBS) with 250uL 100nM DTT in 4% TEA
    • React for 10 minutes
    • Extract DTT with 4 2mL aliquots of ethyl acetate (DTT will partition into ethyl acetate, DNA will partition into aqueous solution)
  • Combine 231.3uL 2.226uM DNA, 231.3uL 29.67nM 14.7nm AuNPs, and 37.3uL sodium citrate buffer prepared 07/11/2013
    • Final Concentrations in Solution:
      • DNA: 1.03uM
      • AuNP: 13.73nM
      • Sodium citrate: 20mM
  • React for 10 minutes
  • Centrifuge 4 times, extracting the supernatant each time and then redispersing in 250uL 50nM HEPES buffer
  • Take absorbance of final solution and three supernatants

Notes

Procedure stopped because the nanoparticles could not be extracted by centrifugation. Absorbance data to be taken tomorrow.

Procedure Update: Silica-coating AuNPs by MHA

We decided to follow the MHA procedure because MHA reduces the number of gold cores per silica-coated particles, and we would ideally have each individual gold nanoparticle covered by a layer of silica. Using the MHA versus not using it has also been very effective in the trials conducted so far, and we think that using MHA will allow us to reach our desired optimal shell thickness of approximately 35nm.

  • Originally detailed on 05/24/2013
  • Day One:
    • Attach MHA to AuNPs: add 5mL of 10mM MHA to 5mL of AuNPs, stir overnight
      • To prepare 10mM MHA solution:
      • (5 mL)×(L/1000mL)×(10mmoles/L)×(1mol/1000mmoles)×(288.49g/mol)=0.0144g MHA in 5mL 2-propanol
      • To prepare 5mL of 9.4nM AuNPs:
      • (29.67nM)V=(9.4nM)(5mL)
      • V=1.584mL AuNPs in 3.416mL PBS buffer
      • Combine MHA and AuNP solutions
  • Day Two:
    • Prepare the following solutions:
  1. 1mL 9.4nM, 10nm AuNP&MHA/20μL TES/0.125mL NH4OH/5mL propanol
  2. 1mL 9.4nM, 10nm AuNP&MHA/15μL TES/0.125mL NH4OH/5mL propanol
  3. 1mL 9.4nM, 10nm AuNP&MHA/10μL TES/0.125mL NH4OH/5mL propanol
  4. 1mL 9.4nM, 10nm AuNP&MHA/5μL TES/0.125mL NH4OH/5mL propanol
    • 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)



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