User:Allison K. Alix/Notebook/Thesis Research/2013/04/15: Difference between revisions

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5. React for 10 minutes
5. React for 10 minutes


6. Centrifuge 4 times, each time re-dispersing in HEPES buffer
6. Centrifuge 4 times, each time re-dispersing in 500 μL 50mM HEPES buffer


7. Save all supernatants and run Absorbance/Fluorescence Measurements on all
7. Save all supernatants and run Absorbance/Fluorescence Measurements on all

Revision as of 10:17, 15 April 2013

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Objectives

  • Repeat Gel Electrophoresis on same samples as 4/12/13 (Stained in ethidium bromide for too long
  • Bind AuNP to DNA-thiol hybrid

Procedures

Part 1: Repeating Gel Electrophoresis

  • Prepare samples the same way

Part 2: Reacting AuNP with DNA-thiol

1. Mix 250μL 2.5μM ThT/727.5nM thiol-DNA synthesized 04/05/2013 with 250μL 4% TEA containing 100mM DTT

  • New DNA concentration: 363.75nM, New ThT Concentration: 1.25μM

2. React 10 minutes

3. Extract DTT using 4 2mL aliquots of ethyl acetate. DTT partitions into ethyl acetate and DNA partitions into aqueous phase

4. Mix 232.4μL 1.25μM ThT/363.75nM thiol-DNA with 232.4μL 4.85nM AuNP and 35.2μL sodium citrate buffer

  • Original concentration AuNP 19.4nM, concentration of solution needs to be 1:75 AuNP:DNA

363.75nM/74 = 4.85nM

19.4nM (x) = 4.85nM (250μL)

x = 62.5μL AuNP in 187.5μL flitered water

5. React for 10 minutes

6. Centrifuge 4 times, each time re-dispersing in 500 μL 50mM HEPES buffer

7. Save all supernatants and run Absorbance/Fluorescence Measurements on all


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