User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/05/29

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May 29, 2013

Reacting thiol DNA with ThT

Procedure

from Allison Alix 04/05/2013<br.>

  • Combine 200μL of 5μM ThT (from AA 04/02/13) and 200μL 1.455μM thiol DNA

new ThT concentration in solution:<br.> 5μM(200×10-6L)=0.001μmoles<br.> 0.001μmoles/(400×10-6L)=2.5μM<br.> <br.> thiol DNA concentration to 1.455 from 53.1:<br.> (53.1μM)V=(1.455μM)(200μL)<br.> V=5.48μL 53.1μM thiol DNA in 194μL filtered water(?)<br.> <br.> new thiol DNA concentration in solution:<br.> 1.455μM(200×10-6L)=2.91×10-4μmoles<br.> 2.91×10-4μmoles/(400×10-6L)=0.7275μM=727.5nM<br.> <br.>

  • Heat at ~75°C for 25 minutes
  • Allow solution to cool to room temperature
  • Take absorbance and florescence measurements

Data

Absorbance

Peak observed at 414.00nm 05292013 corrected thiol DNA and ThT absorbance.png

Florescence

Excited at 414nm; peak observed ~500nm Thiol DNA and ThT florescence.png

Reacting AuNPs with thiol DNA/ThT Solution

Procedure

from Allison Alix 04/15/2013<br.> re-do of 05/28/2013<br.>

  • Combined 250μL 2.5μM ThT/727.5nM thiol DNA with 250μL 4% TEA with 100mM DTT, both made 05/29/2013

new DNA concentration: 727.5nM(250×10-6L)=0.181875nmoles<br.> 0.181875nmoles/(500×10-6L)=363.75nM<br.> new ThT concentration:<br.> 2.5μM(250×10-6L)=0.000625<br.> 0.000625/(500×10-6L)=1.25μM<br.>

  • Allowed to react for 10 minutes
  • Extracted DTT with 4, 2mL aliquots of ethyl acetate, removing the ethyl acetate layer between each aliquot
    • DTT dissolved in ethyl acetate layer (top)<br.>
  • Combined thiol DNA/ThT and TEA with AuNPs and citrate buffer
    • 1 AuNP:75 DNA ratio in solution<br.>
    • Combined 232.4μL 1.25μM ThT/363.75nM thiol-DNA with 232.4μL 4.85nM AuNP and 35.2μL sodium citrate buffer
  • Allowed to react for 10 minutes
  • Redispersed in 500μL 50mM HEPES buffer before each of 4 rounds of centrifuging
    • First round: centrifuged at 10000rpm for 14 minutes, dispered in 200μL HEPES buffer, then 12000rpm for 7 minutes
      • Removed top layer (since no separation observed), redispersed in 300μL HEPES buffer
    • Second round: centrifuged at 12000rpm for 12 minutes
      • Removed top layer (since no separation observed), redispersed in 300μL HEPES buffer
    • Third round: centrifuged at 12000rpm for 12 minutes
  • Saved all supernatants

Data

Absorbance

Thiol DNA, ThT and AuNP.png<br.> Dark Blue: Water<br.> Light Blue: Thiol DNA, ThT, AuNP solution<br.> Red: Supernatant 1<br.> Green: Supernatant 2<br.> Purple: Supernatant 3<br.>

Observations

Again, the extracted sample did not separate in the centrifuge as expected. I centrifuged as usual (though with larger time intervals and higher speeds) and removed the top "supernantant" layer despite the lack of separation. I took absorbance measurements, with inconclusive results.