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

(Difference between revisions)
 Revision as of 14:40, 3 June 2013 (view source) (→Sample Preparation for FCS and Calculations)← Previous diff Revision as of 14:40, 3 June 2013 (view source) (→MB Solution Dilutions)Next diff → Line 83: Line 83: **Thiol DNA- 40.26nM **Thiol DNA- 40.26nM =====MB Solution Dilutions===== =====MB Solution Dilutions===== - 7.140.33nM MB + 7. 140.33nM MB 3.32μL in 26.68μL PBS buffer 3.32μL in 26.68μL PBS buffer - 8.70.165nM MB + + 8. 70.165nM MB 15μL 140.33nM MB in 15μL PBS buffer 15μL 140.33nM MB in 15μL PBS buffer - 9.14.033nM MB + + 9. 14.033nM MB 6μL 70.165nM MB in 24μL PBS buffer 6μL 70.165nM MB in 24μL PBS buffer

## Revision as of 14:40, 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

#### 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