User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/03

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*ThT- 2.2245μM
*ThT- 2.2245μM
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=====Absorbance and Fluorescence Data=====
[[Image:Abs_data_thiol_DNA,_AuNP,_MB_06032013.png]]
[[Image:Abs_data_thiol_DNA,_AuNP,_MB_06032013.png]]
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Revision as of 10:10, 8 July 2013

Measurements and Sample Prep for FCS Main project page
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June 3, 2013

Absorbance and Fluorescence Data

Finish preparing thiol DNA, AuNP and ThT sample

2[ThT]:1[DNA]→ 2.2245μM ThT
10% of 250μL=25μL (within 10% allows for accurate concentration comparison)
Total solution volume: 275μL
(2.2245×10-6M)(275×10-6L)=6.11738×10-10moles ThT
(6.11738×10-10moles)/(25×10-6L)=24.5μM ThT

To make 24.5μM ThT:
(120μM)V=(24.5μM)(25μL)
V=5.1μL in 19.9μL PBS buffer

Add 25μL of 24.5μM ThT to 250μL DNA/AuNP solution
New concentrations:

  • thiol DNA- 1.011μM
  • AuNP- 13.486μM
  • ThT- 2.2245μM


Absorbance and Fluorescence Data

Image:Abs_data_thiol_DNA,_AuNP,_MB_06032013.png

Image:Abs_data_thiol_DNA,_ThT_06032013.png

Image:Fluor_data_thiol_DNA,_AuNP,_MB_06032013.png

Image:Fluor_data_thiol_DNA,_AuNP,_ThT_06032013.png

Notes

Samples containing Molecular Beacon excited at 532nm
Samples containing ThT excited at 440nm

Sample Preparation for FCS and Calculations

Thiol DNA, MB, AuNP Solution Dilutions
  • Starting Concentrations:
    • AuNP- 9.03nM
    • MB-1268nM
    • Thiol DNA- 677.157nM

1. 1nM AuNPs
(9.03nM)V=(1nM)(30μL)
V=3.32μL in 26.68μL PBS buffer

  • New Concentrations:
    • AuNP- 1nM
    • MB- 140.33nM
    • Thiol DNA- 74.94nM

2. 500pM AuNPs
15μL 1nM solution in 15μL PBS buffer

  • New Concentrations:
    • AuNP- 500pM
    • MB- 70.165nM
    • Thiol DNA- 37.47nM

3. 100pM AuNPs
6μL 500pM solution in 24μL PBS buffer

  • New Concentrations:
    • AuNP- 100pM
    • MB- 14.033nM
    • Thiol DNA- 7.55nM
Thiol DNA, MB Solution Dilutions
  • Starting Concentrations:
    • MB- 1268nM
    • Thiol DNA- 727.5nM

4. 140.33nM MB
3.32μL in 26.68μL PBS buffer

  • New Concentrations:
    • MB- 140.33nM
    • 80.51nM

5. 70.165nM MB
15μL 140.33nM MB in 15μL PBS buffer

  • New Concentrations:
    • MB- 70.165nM
    • Thiol DNA- 40.26nM

6. 14.033nM MB
6μL 70.165nM MB in 24μL PBS buffer

  • New Concentrations:
    • MB- 14.033nM
    • Thiol DNA- 40.26nM
MB Solution Dilutions
  • Starting Concentrations:
    • MB- 1268nM

7. 140.33nM MB
3.32μL in 26.68μL PBS buffer

8. 70.165nM MB
15μL 140.33nM MB in 15μL PBS buffer

9. 14.033nM MB
6μL 70.165nM MB in 24μL PBS buffer



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