User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/04/02: Difference between revisions

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==Objectives==
==April 2, 2013==
=====Hybridize thiol DNA and Molecular Beacon, then AuNP=====
DNA: 53.1μM → 727.5nM
<br.>
(53.1μM)V=(500μL)(1455nM)<br.>
V=13.7μL 53.1μM DNA in 486.3μL filtered H<sub>2</sub>O <br.>
(1455nM)V=(200μL)(727.5nM)<br.>
V=100μL 1455nM DNA in 100μL H<sub>2</sub>O <br.>


* Bind DNA to MB and AuNP
MB: 7.31μM → 800nM<br.>


* React AuNP with ethanethiol
(7130nM)V=(800nM)(50μL)<br.>
V=5.61μL 7.31μM MB in 44.53μL H<sub>2</sub>O<br.>
<br.>
Combine 50μL 727.5nM thiol DNA with 50μL 800nM MB, heat at 75°C for 25 minutes, cool to room temperature.<br.>


==Procedures==
=====Hybridize thiol DNA and ThT, then AuNP=====
ThT: from January 29, 2013- assumed 100%, Dr. Miller's 5mM ThT used in this procedure<br.>
<br.>
(5mM)V=(5μM)(10mL)<br.>
V= 10μL of 5mM ThT in 9990μL PBS buffer<br.>
(5μM)V=(800nM)(1mL)<br.>
V= 160μL 5μM ThT in 840μL PBS buffer<br.>
<br.>
Combine 50μL 727.5nM thiol DNA with 50μL 800nM ThT<br.>
 
=====AuNP with Ethanethiol=====
AuNP radius: (from Dr. Miller March 1, 2013) 14nm diameter=7nm radius<br.>
Area of AuNP: 4πr<sup>2</sup>=615.75 nm<sup>2</sup><br.>
Sulfur atom radius: 200pm diameter=100pm radius<br.>
Area of sulfur atom: r<sup>2</sup>=40000pm<sup>2</sup><br.>
sulfur atoms/AuNP=(6.1575×10<sup>-16</sup>m<sup>2</sup>)/(4×10<sup>-20</sup>m<sup>2</sup>)=15393<br.>
<br.>
AuNP: 19.4nM<br.>
Ethanethiol: make 298μM, density = 0.839g/mL<br.>
<br.>
2.5μL × (10<sup>-6</sup>L/1μL) × (1000mL/1L) × (0.839g/1mL) × (1 mol/ 62.134g) = 3.376×10<sup>-4</sup>mol = 0.34 M = 340 mM<br.>
<br.>
(1mM)V=(340mM)(1mL)<br.>




Revision as of 13:21, 12 July 2013

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April 2, 2013

Hybridize thiol DNA and Molecular Beacon, then AuNP

DNA: 53.1μM → 727.5nM <br.> (53.1μM)V=(500μL)(1455nM)<br.> V=13.7μL 53.1μM DNA in 486.3μL filtered H2O <br.> (1455nM)V=(200μL)(727.5nM)<br.> V=100μL 1455nM DNA in 100μL H2O <br.>

MB: 7.31μM → 800nM<br.>

(7130nM)V=(800nM)(50μL)<br.> V=5.61μL 7.31μM MB in 44.53μL H2O<br.> <br.> Combine 50μL 727.5nM thiol DNA with 50μL 800nM MB, heat at 75°C for 25 minutes, cool to room temperature.<br.>

Hybridize thiol DNA and ThT, then AuNP

ThT: from January 29, 2013- assumed 100%, Dr. Miller's 5mM ThT used in this procedure<br.> <br.> (5mM)V=(5μM)(10mL)<br.> V= 10μL of 5mM ThT in 9990μL PBS buffer<br.> (5μM)V=(800nM)(1mL)<br.> V= 160μL 5μM ThT in 840μL PBS buffer<br.> <br.> Combine 50μL 727.5nM thiol DNA with 50μL 800nM ThT<br.>

AuNP with Ethanethiol

AuNP radius: (from Dr. Miller March 1, 2013) 14nm diameter=7nm radius<br.> Area of AuNP: 4πr2=615.75 nm2<br.> Sulfur atom radius: 200pm diameter=100pm radius<br.> Area of sulfur atom: r2=40000pm2<br.> sulfur atoms/AuNP=(6.1575×10-16m2)/(4×10-20m2)=15393<br.> <br.> AuNP: 19.4nM<br.> Ethanethiol: make 298μM, density = 0.839g/mL<br.> <br.> 2.5μL × (10-6L/1μL) × (1000mL/1L) × (0.839g/1mL) × (1 mol/ 62.134g) = 3.376×10-4mol = 0.34 M = 340 mM<br.> <br.> (1mM)V=(340mM)(1mL)<br.>



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