User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/05/28

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May 28, 2013

Reacting AuNPs with thiol DNA

Procedure

from Allison Alix 04/15/2013<br.>

  • Combine 80μL 2.5μM ThT/727.5nM thiol DNA with 80μL 4% TEA with 100mM DTT<br.>

new DNA concentration:<br.> 727.5nM(80×10-6L)=0.0582nmoles <br.> 0.0582nmoles/(160×10-6L)=363.75nM<br.> <br.> new ThT concentration:<br.> 2.5μM(80×10-6L)=0.0002<br.> 0.0002/(160×10-6L)=1.25μM<br.>

  • Allowed to react for 10 minutes<br.>
  • Extracted DTT with 4, 2mL aliquots of ethyl acetate, removing the ethyl acetate layer between each aliquot<br.>

DTT dissolved in ethyl acetate layer (top)<br.>

  • Combine thiol DNA/ThT and TEA with AuNPs and citrate buffer<br.>

8μL thiol DNA/ThT w/TEA from extraction, add 92μL filtered water to make 100μL<br.> new DNA concentration:<br.> 363.75nM(80×10-6L)=0.00291nmoles<br.> 0.00291nmoles/(100×10-6L)=29.1nM<br.> <br.> 29.1nM/75=0.4nM AuNP for 1 AuNP:75 DNA ratio in solution<br.> (19.4nM)V=(0.4nM)(100μL)<br.> V=0.5μL AuNPs in 99.5μL filtered water<br.>

  • Combine 100μL 291.nM thiol DNA/0.1μM ThT with 100μL 0.4nM AuNPs and 50μL sodium citrate buffer<br.>
  • Allowed to react for 10 minutes<br.>
  • Redisperse in 250μL 50mM HEPES buffer<br.>
  • Centrifuge for 25 minutes at 10000 rpm<br.>

Observations

Since the extracted sample did not separate in the centrifuge, I did not centrifuge 4 times and take absorbance/florescence measurements, as was done previously. I will attempt to run the procedure again tomorrow either with a different thiol DNA/Tht solution, or after synthesizing more of the thiol DNA/ThT solution.

Error

During the extraction process, after the last aliquot of ethyl acetate, the solution failed to (visibly) separate as it had for each other aliquot/separation procedures. I attempted to remove the ethyl acetate/DTT component as usual but since the sample did not separate when centrifuged, there was not a significant amount of thiol DNA/ThT/4% TEA solution in the sample. I was unable to identify the date on which the thiol DNA/ThT solution I started with was synthesized, so the sample may have been at a different concentration, been contaminated, or possibly too old. Ideally, I should have synthesized a fresh solution to confirm the date and component concentrations.