User:Allison K. Alix/Notebook/Thesis Research/2013/05/31

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Objectives

  • Prepare an AuNP/DNA sample
  • Prepare ThT/DNA sample
  • Prepare MB sample
  • Prepare ThT sample
  • Prepare 0.05M phosphate buffer

Procedures

Part 1: Preparation of AuNP-DNA sample

Final concentrations of AuNP and DNA should be 14.835nM and 1.11625μM respectively

1) 53.1μM(x)=(4.4506μM)(250μL)

x= 20.95μL in 229.05μL PBS

mix 20.95μL 53.1μM thiol-DNa with 229.05μL PBS

2) Mix 250μL DNA solution with 250μL 100mM DTT in 4% TEA

NEW [DNA] = 2.225μM

3) React 10 minutes

5) Extract DTT from solution using 4 2mL aliquots of ethyl acetate

6) Mix 232.4μL DNA, 232.4μL 29.7nM AuNP, and 36.2μL sodium citrate buffer

NEW [DNA] = 1.034μM NEW {[AuNP] = 13.805nM

7) React 10 minutes

8) Centrifuge for 7 minutes at 10000rpm

Part 2: Preparation of thiol-DNA/ThT solution

concentration of DNA must match [DNA] in AuNP/DNA solution and [ThT] must be at least twice this

1) 5.82μM(x)=(2.2253μM)(250μL)

x = 95.6μL in 154.4μL PBS to make 250μL 2.2263μM DNA

2) 120μM(x)=(250μL)(4.44μM)

x = 9.25μL in 240.75μL PBS to make 250μL 4.44μM ThT

3) Mix 250μL 2.2263μM DNA with 250μL 4.44μM ThT

NEW [DNA] = 1.11265μM NEW [ThT] = 2.22μM

Part 3: Preparation of MB

1) 7.61μM(x)=(100μL)(1.268μM)

x = 16.7μL 7.61μM MB in 83.3μL PBS to make 100μL 1.268μM MB

Part 4: Preparation of ThT

concentration of ThT must match [ThT] in DNA/ThT sample

1) 120μM(x)=(250μL)(2.22μM)

x = 4.625μL in 245.375μL PBS to make 250μL 2.22μM ThT

Part 5: Preparation of 0.05M phosphate buffer, pH=7.4

*to be used to attach DNA to silica coated AuNP

[Base] = potassium phosphate, monobasic = 136.09 g/mol

[Acid] = potassium phosphate, dibasic = 174.20 g/mol

pH = pKa + log(Base)/(Acid)

7.4 - 6.8 = log(Base)/(Acid)

Base/Acid = 3.98

Acid + Base = 0.05M

Base= 0.05M-Acid

(0.05M-Acid)/Acid = 3.98

[Acid] = 0.0100M = 1.742g

[Base] = 0.0400M = 5.4436g

  • NOTE that this is based on 1L of buffer being used and only 500mL was actually prepared. One half of the masses above were added to ~400mL of water. The pH was adjusted to 7.4 using ~2M NaOH and the remaining water was then added.





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