User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/05/31

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May 31, 2013

Absorbance and Florescence Measurements

Absorbance 05312013.png <br.> <br.> Fluorescence 05312013.png<br.>

Concentrations
  • thiol DNA- 615.59nM<br.>
  • AuNP- 9.03nM<br.>
  • Molecular Beacon- 1268nM<br.>

Notes

Samples excited at 532nm, fluorescence peak observed at approximately 565nm

Sample Preparation

Molecular Beacon, final concentration: 1268nM

(7.61μM)V=(1.268μM)(100μL) V=16.7μL 7.61μM MB in 83.3μL PBS buffer

AuNP and thiol DNA, final concentrations: 14.835μM and 1.11265μM

(53.1μM)V=(4.45μM)(250μL)<br.> V=20.95μL 53.1μM thiol DNA in 229.05μL PBS buffer<br.>

  • thiol DNA extraction following the same method from Allison Alix on 05/15/2013 without ThT
  • Combine 232μL 29.67nM, 10nm AuNP, 232μL extracted thiol DNA, and 36μL sodium citrate buffer
    • Always add sodium citrate buffer last, to avoid disruption of DNA by the change in pH
  • To be continued on Monday, June 3, 2013
    • Try to sonicate AuNPs
ThT and thiol DNA, final concentrations: 2.2244μM and 1.011μM

(53.1μM)V=(2.022μM)(250μL)<br.> V=55.6μL 53.1μM thiol DNA in 194.38μL PBS buffer<br.> (20μM)V=(4.45μM)(250μL)<br.> V=55.6μL 20μM ThT in 194.38μL PBS buffer<br.>

ThT, final concentration: 2.2245μM

(20μM)V=(2.2245μM)(250μL)<br.> V=27.8μL 20μM ThT in 222.2μL PBS buffer<br.>

Notes

  • Finish preparing AuNP and thiol DNA sample
  • Run absorbance and fluorescence for all four samples
  • Compile data into one absorbance graph and one fluorescence graph
  • Eventually run samples on FCS
  • Next week: FCS, silica-coating on nanoparticles