User:Allison K. Alix/Notebook/Thesis Research/2013/05/14

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==Observations==
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In the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point.

Revision as of 12:22, 14 May 2013

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Objectives

  • React thiol-DNA/ThT/AuNP at 150:1 concentrations of thiol-DNA:AuNP
  • Run 2% agarose gel on thiol-DNA/ThT/AuNP synthesized on 04/23/2013
  • Take absorbance and fluorescence measurements of thiol-DNA/ThT before and after attaching AuNP

Procedures

  • New AuNP synthesized is 29.67nM with a diameter of 10nm

Previous concentrations used:

Initial Final
thiol-DNA 5.82μM 727.5nM
ThT 5μM 625nM
AuNP 19.4nM 9.7nM

To be used:

Initial Final
thiol-DNA 17.8μM 2.225μM
ThT 20μM 2.5μM
AuNP 29.67nM 14.835nM

Calculations:

Preparing 17.8μM thiol-DNA

original concentration of thiol-DNA- 53.1μM

53.1μM (x) = (17.8μM)(250μL) x= 83.8μL in 166.2μL buffer

Preparing 20μM ThT

original conc ThT= 5mM

5mM (x) = (0.02mM)(10mL) x=40μL in 9960μL H2O


Part 1: hybridizing thiol-DNA with ThT

1) Mix 250μL 17.8μM thiol-DNA with 250μL 20μM ThT

  • NEW concentrations: 8.9μM thiol-DNA and 10μM ThT

2) Heat at 75°C for ~25min

3) Cool to room temperature

4) Take UV-Vis/Fluorescence measurements of thiol-DNA/ThT sample

Part 2: Hybridizing thiol-DNA/ThT with AuNP

1) Mix 250 μL 8.9μM thiol-DNA/10μM ThT solution with 250μL 4%TEA containing 100mM DTT

  • NEW concentrations: 4.45μM thiol-DNA/5μM ThT

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

3) Mix 232.4μL of thiol-DNA/ThT with 232.4μL 29.67nM AuNP and 35.2μL sodium citrate buffer

  • NEW concentrations: 2.06μM thiol DNA, 2.324μM ThT, 13.79nM AuNP

Part 3: Gel Electrophoresis (2% Agarose)

Prepare the following:

1) 5μL 100bp ladder with 1μL loading buffer

2) 5μL thiol-DNA/ThT/AuNP with 1μL loading buffer

3) 5μL supernatant 1 with 1μL loading buffer

4) 5μL supernatant 2 with 1μL loading buffer

5) 5μL supernatant 3 with 1μL loading buffer

Data

Image:Pic_of_abs.png


Image:Pic_of_fluor.png


Observations

In the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point.




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