User:Allison K. Alix/Notebook/Thesis Research/2013/05/15

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Objectives

  • Add more concentrated ThT to DNA/ThT sample to ensure [ThT] is double that of [DNA]
  • Attach AuNP to DNA/ThT Sample
  • Take UV-Vis/Fluorescence of DNA/ThT/AuNP Sample

Procedures

  • Currently, the DNA/ThT mixture is 8.901μM DNA and 10μM ThT. Because there are 8 telomerase base repeats on the thiol DNA, we want to make sure there is twice as much ThT in solution as there is DNA so that two quadruplexes may be formed. Instead of making a new solution, we can simply add more ThT so the new ThT concentration is now 20μM. We want to add as little as possible as to not disturb the concentration of DNA by too much (10% or 50μL since we are starting with a solution of 500μL)

Calculations:

10x10-6mol/L x 500x10-6L = 5x10-9 moles ThT currently in solution

20x10-6mol/L x 550x10-6L = 1.1x10-8 moles ThT that should be in solution

1.1x10-8 moles - 5x10-9 moles = 6x10-9 moles ThT that need to be added

6x10-9moles/50x10-6L = 1.2x10-4M

  • Add 50μL of 1.2x10-4M ThT to solution to make 20μM ThT

Preparing 0.12mM ThT

5mM (x) = (0.12mM)(500μL)

x = 12μL in 488μL PBS buffer

NEW Concentrations:

ThT: 20μM

thiol-DNA: (8.9μM)(500μL) = x (550μL)

x = 8.09μM

Part 1: Attaching AuNP to thiol-DNA/ThT

1) Add 250μL thiol-DNA/ThT solution to 250μL 4% TEA/100mM DTT solution

NEW Concentrations: 10μM ThT, 4.045μM thiol DNA

2) React 10 minutes

3) Extract DTT using 4 2mL aliquots of ethyl acetate

4) Mix 232.4μL thiol-DNA/ThT with 232.4μL 29.67nM AuNP and 35.2μL 284mM sodium citrate buffer

5) React 10 minutes

NEW concentrations: 5μM ThT, 2.0225μM thiol-DNA, 14.835nM AuNP, 20mM sodium citrate

6) Centrifuge and redisperse in HEPES buffer

Data

Image:Fluor_pic.png




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