User:Allison K. Alix/Notebook/Thesis Research/2013/04/02: Difference between revisions

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{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Reactions of DNA with ThT and MB</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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'''Part 1: Reacting DNA with MB first, followed by a reaction of the DNA-MB hybrid with AuNP'''
'''Part 1: Reacting DNA with MB first, followed by a reaction of the DNA-MB hybrid with AuNP'''


1) Prepare 727.5nm 8-repeat DNA-thiol and 800nm MB
1) Prepare 727.5nM 8-repeat DNA-thiol and 800nM MB


a) 727.5nm 8-repeat DNA-thiol
a) 727.5nM 8-repeat DNA-thiol


(Original concentration of DNA is 53.1μM)
(Original concentration of DNA is 53.1μM)
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x= 13.7μL in 486.3μL H<sub>2</sub>O
x= 13.7μL in 486.3μL H<sub>2</sub>O


1455nM(x) = 727.5nm(200μL)
1455nM(x) = 727.5nM(200μL)


x= 100μL 1455nM in 100μL H<sub>2</sub>O
x= 100μL 1455nM in 100μL H<sub>2</sub>O
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6.1575x10<sup>-16</sup>m<sup>2</sup>/4x10<sup>-20</sup>m<sup>2</sup> = 15393 Sulfur atoms for every 1 AuNP
6.1575x10<sup>-16</sup>m<sup>2</sup>/4x10<sup>-20</sup>m<sup>2</sup> = 15393 Sulfur atoms for every 1 AuNP
2) Prepare 1mL ethanethiol solution
*original concentrationof AuNP = 19.4nM
desired concentration of ethanethiol = 19.4nM x(15393) = 298624.2nM = 298μM
density of ethanethiol = 0.839g/mL
298x10<sup>-6</sup>mol/L




Revision as of 08:14, 12 April 2013

Reactions of DNA with ThT and MB <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Objectives

  • Bind DNA to MB and AuNP
  • React AuNP with ethanethiol

Procedures

Part 1: Reacting DNA with MB first, followed by a reaction of the DNA-MB hybrid with AuNP

1) Prepare 727.5nM 8-repeat DNA-thiol and 800nM MB

a) 727.5nM 8-repeat DNA-thiol

(Original concentration of DNA is 53.1μM)

53.1μM(x)=(500μL)(1455nm)

x= 13.7μL in 486.3μL H2O

1455nM(x) = 727.5nM(200μL)

x= 100μL 1455nM in 100μL H2O

b) Prepare 800nm MB

(Original concentration of MB is 7.31μM)

7130nm(x) = (800nM)(50μL)

x = 5.47μL 7.31μM in 44.53 μL H2O

3) Mix 50μL 727.5nM DNA with 50μL 800nM MB

4) Hybridize by placing mixture on heat block @ 76°C for ~25minutes

5) Turn off heat block and allow cooling to room temperature

Part 2: Reacting DNA with ThT first, followed by a reaction of the DNA-ThT hybrid with AuNP

1) Prepare 727.5nM DNA (see Part 1, step 1a)

2) Prepare 800nM ThT

  • Note that the ThT prepared on 01/29/2013 assumed a 100% mass, when it was actually 75%. Use a 5mM ThT solution prepared by Dr. Miller.

5mM(x) = (5μM)(10mL)

10μL of 5mM ThT in 9990μL buffer

5μM (x) = (800nm)(1mL)

x= 160μL 5μM ThT in 840μL buffer

3) Mix 50μL 727.5nM DNA with 50μL 800nM ThT

Part 3: React AuNP's with ethanethiol

1) Calculate how many sulfur atoms to cover 1 nanoparticle

  • Diameter of AuNP (synthesized 3/1/13 by Dr. Miller) is 14nm, so radius, r=7nm
  • A = 4π(r2)= 4π(7x10-9)2= 6.1575x10-16m2
  • atomic radius of 1 sulfur atom = 100pm so, diameter of 1 S = 200pm

(200x10-12m)2 = 4x10-20m2

6.1575x10-16m2/4x10-20m2 = 15393 Sulfur atoms for every 1 AuNP

2) Prepare 1mL ethanethiol solution

  • original concentrationof AuNP = 19.4nM

desired concentration of ethanethiol = 19.4nM x(15393) = 298624.2nM = 298μM

density of ethanethiol = 0.839g/mL

298x10-6mol/L










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