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

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(Sample Preparation for FCS and Calculations)
(MB Solution Dilutions)
Line 83: Line 83:
**Thiol DNA- 40.26nM
**Thiol DNA- 40.26nM
=====MB Solution Dilutions=====
=====MB Solution Dilutions=====
-
7.140.33nM MB<br.>
+
7. 140.33nM MB<br.>
3.32μL in 26.68μL PBS buffer<br.>
3.32μL in 26.68μL PBS buffer<br.>
-
8.70.165nM MB<br.>
+
<br.>
 +
8. 70.165nM MB<br.>
15μL 140.33nM MB in 15μL PBS buffer<br.>
15μL 140.33nM MB in 15μL PBS buffer<br.>
-
9.14.033nM MB<br.>
+
<br.>
 +
9. 14.033nM MB<br.>
6μL 70.165nM MB in 24μL PBS buffer<br.>
6μL 70.165nM MB in 24μL PBS buffer<br.>

Revision as of 13:40, 3 June 2013

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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


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

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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