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

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(June 3, 2013)
Current revision (10:10, 8 July 2013) (view source)
(Absorbance and Fluorescence Data)
 
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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Measurements and Sample Prep for FCS</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==June 3, 2013==
==June 3, 2013==
===Absorbance and Fluorescence Data===
===Absorbance and Fluorescence Data===
-
Finish preparing thiol DNA, AuNP and ThT sample<br.>
+
=====Finish preparing thiol DNA, AuNP and ThT sample=====
2[ThT]:1[DNA]→ 2.2245μM ThT<br.>
2[ThT]:1[DNA]→ 2.2245μM ThT<br.>
10% of 250μL=25μL (within 10% allows for accurate concentration comparison)<br.>
10% of 250μL=25μL (within 10% allows for accurate concentration comparison)<br.>
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*ThT- 2.2245μM
*ThT- 2.2245μM
<br.>
<br.>
 +
=====Data=====
[[Image:Abs_data_thiol_DNA,_AuNP,_MB_06032013.png]]
[[Image:Abs_data_thiol_DNA,_AuNP,_MB_06032013.png]]
<br.>
<br.>
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<br.>
<br.>
[[Image:Fluor_data_thiol_DNA,_AuNP,_ThT_06032013.png]]
[[Image:Fluor_data_thiol_DNA,_AuNP,_ThT_06032013.png]]
 +
====Notes====
====Notes====
Samples containing Molecular Beacon excited at 532nm<br.>
Samples containing Molecular Beacon excited at 532nm<br.>
Samples containing ThT excited at 440nm<br.>
Samples containing ThT excited at 440nm<br.>
-
===Sample Preparation for FCS and Calculations===
 
 +
===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<br.>
 +
(9.03nM)V=(1nM)(30μL)<br.>
 +
V=3.32μL in 26.68μL PBS buffer
 +
*New Concentrations:
 +
**AuNP- 1nM
 +
**MB- 140.33nM
 +
**Thiol DNA- 74.94nM
 +
2. 500pM AuNPs<br.>
 +
15μL 1nM solution in 15μL PBS buffer
 +
*New Concentrations:
 +
**AuNP- 500pM
 +
**MB- 70.165nM
 +
**Thiol DNA- 37.47nM
 +
3. 100pM AuNPs<br.>
 +
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<br.>
 +
3.32μL in 26.68μL PBS buffer
 +
*New Concentrations:
 +
**MB- 140.33nM
 +
**80.51nM
 +
5. 70.165nM MB<br.>
 +
15μL 140.33nM MB in 15μL PBS buffer
 +
*New Concentrations:
 +
**MB- 70.165nM
 +
**Thiol DNA- 40.26nM
 +
6. 14.033nM MB<br.>
 +
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<br.>
 +
3.32μL in 26.68μL PBS buffer<br.>
 +
<br.>
 +
8. 70.165nM MB<br.>
 +
15μL 140.33nM MB in 15μL PBS buffer<br.>
 +
<br.>
 +
9. 14.033nM MB<br.>
 +
6μL 70.165nM MB in 24μL PBS buffer<br.>

Current revision

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


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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