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

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

new ThT concentration in solution:
5μM(200×10-6L)=0.001μmoles
0.001μmoles/(400×10-6L)=2.5μM

thiol DNA concentration to 1.455 from 53.1:
(53.1μM)V=(1.455μM)(200μL)
V=5.48μL 53.1μM thiol DNA in 194μL filtered water(?)

new thiol DNA concentration in solution:
1.455μM(200×10-6L)=2.91×10-4μmoles
2.91×10-4μmoles/(400×10-6L)=0.7275μM=727.5nM

  • 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 Image:05292013_corrected_thiol_DNA_and_ThT_absorbance.png

Florescence

Excited at 414nm; peak observed ~500nm Image:Thiol_DNA_and_ThT_florescence.png

Reacting AuNPs with thiol DNA/ThT Solution

Procedure

from Allison Alix 04/15/2013
re-do of 05/28/2013

  • 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
0.181875nmoles/(500×10-6L)=363.75nM
new ThT concentration:
2.5μM(250×10-6L)=0.000625
0.000625/(500×10-6L)=1.25μM

  • 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)
  • Combined thiol DNA/ThT and TEA with AuNPs and citrate buffer
    • 1 AuNP:75 DNA ratio in solution
    • 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

Image:Thiol_DNA,_ThT_and_AuNP.png
Dark Blue: Water
Light Blue: Thiol DNA, ThT, AuNP solution
Red: Supernatant 1
Green: Supernatant 2
Purple: Supernatant 3

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.


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