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

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(Procedures)
Current revision (13:17, 15 April 2013) (view source)
(Procedures)
 
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==Entry title==
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==Objectives==
* Repeat Gel Electrophoresis on same samples as 4/12/13 (Stained in ethidium bromide for too long
* Repeat Gel Electrophoresis on same samples as 4/12/13 (Stained in ethidium bromide for too long
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==Procedures==
==Procedures==
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Part 1: Repeating Gel Electrophoresis
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'''Part 1: Repeating Gel Electrophoresis'''
* Use same procedure as [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/11|04/11/2013]]
* Use same procedure as [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/11|04/11/2013]]
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* Prepare samples the same way
* Prepare samples the same way
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Part 2: Reacting AuNP with DNA-thiol
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'''Part 2: Reacting AuNP with DNA-thiol'''
1. Mix 250μL 2.5μM ThT/727.5nM thiol-DNA synthesized [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/05|04/05/2013]] with 250μL 4% TEA containing 100mM DTT  
1. Mix 250μL 2.5μM ThT/727.5nM thiol-DNA synthesized [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/05|04/05/2013]] with 250μL 4% TEA containing 100mM DTT  
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5. React for 10 minutes
5. React for 10 minutes
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6. Centrifuge 4 times, each time re-dispersing in 500 μL 50mM HEPES buffer
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7. Save all supernatants and run Absorbance/Fluorescence Measurements on all
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Current revision

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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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