User:Allison K. Alix/Notebook/Thesis Research/2013/05/29

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Objectives

  • Prepare DTT/TEA solution to be used in DNA extraction procedures.
  • Prepare DNA/AuNP mixture
  • Bind MB to DNA/AuNP

Procedures

Part 1: Preparation of 100mM DTT in 4% TEA solution

Original DTT concentration 1M

1M(x) = (100x10-3M)(1mL)

x = 0.1mL = 100μL in 900μL 4% TEA

4%(900μL) = 36μL TEA in 864μL PBS

OVERALL: 100μL 1M DTT + 36μL TEA + 864μL PBS

Part 2: Prepare DNA-AuNP to bind MB to

1) Mix 100μL 2.91μM thiol-DNA with 100μL 100mM DTT in 4% TEA. React 10 minutes

[DNA]=1.45μM

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

3) Mix 93μL thiol-DNA with 93μL 19.4nM AuNP and 14μL sodium citrate buffer. React 10 minutes.

[DNA] = 727.5nM [AuNP] = 9.7nM

4) Centrifuge and redisperse in 50mM HEPES buffer

5) Mix 20μL of 7.61μM MB with 100μL of thiol DNA/AuNP mixture

6) Heat @75°C for 25 minutes.

7) Observe absorbance and fluorescence spectra




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