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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Reactions of DNA with ThT and MB</span> | ||
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== | ==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 H<sub>2</sub>O | |||
1455nM(x) = 727.5nM(200μL) | |||
x= 100μL 1455nM in 100μL H<sub>2</sub>O | |||
b) Prepare 800nm MB | |||
(Original concentration of MB is 7.31μM) | |||
7130nm(x) = (800nM)(50μL) | |||
x = 5.47μL 7.31μM in 44.53 μL H<sub>2</sub>O | |||
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 [[User:Allison K. Alix/Notebook/Thesis Research/2013/01/29|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(r<sup>2</sup>)= 4π(7x10<sup>-9</sup>)<sup>2</sup>= 6.1575x10<sup>-16</sup>m<sup>2</sup> | |||
* atomic radius of 1 sulfur atom = 100pm so, diameter of 1 S = 200pm | |||
(200x10<sup>-12</sup>m)<sup>2</sup> = 4x10<sup>-20</sup>m<sup>2</sup> | |||
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
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Objectives
ProceduresPart 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
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
(200x10-12m)2 = 4x10-20m2 6.1575x10-16m2/4x10-20m2 = 15393 Sulfur atoms for every 1 AuNP 2) Prepare 1mL ethanethiol solution
desired concentration of ethanethiol = 19.4nM x(15393) = 298624.2nM = 298μM density of ethanethiol = 0.839g/mL 298x10-6mol/L
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