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

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(Procedures)
Current revision (11:04, 15 May 2013) (view source)
(Observations)
 
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'''Part 2: Hybridizing thiol-DNA/ThT with AuNP'''
'''Part 2: Hybridizing thiol-DNA/ThT with AuNP'''
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1)
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1) Mix 250 μL 8.9μM thiol-DNA/10μM ThT solution with 250μL 4%TEA containing 100mM DTT
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Part 3: Gel Electrophoresis (2% Agarose)
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* NEW concentrations: 4.45μM thiol-DNA/5μM ThT
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2) Extract DTT using 4 2mL aliquots of ethyl acetate
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3) Mix 232.4μL of thiol-DNA/ThT with 232.4μL 29.67nM AuNP and 35.2μL sodium citrate buffer
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* NEW concentrations: 2.06μM thiol DNA, 2.324μM ThT, 13.79nM AuNP
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'''Part 3: Gel Electrophoresis (2% Agarose)'''
Prepare the following:
Prepare the following:
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5) 5μL supernatant 3 with 1μL loading buffer
5) 5μL supernatant 3 with 1μL loading buffer
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==Data==
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[[Image:Pic_of_abs.png]]
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[[Image:Pic_of_fluor.png]]
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[[Image:AliDNAThTAuNPGel.png]]
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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.
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* Note that for the gel electrophoresis, if using the blue/orange loading dye, allow the gel to run until the lightest blue dye is 3/4 of the way across, this should be the dye that travels the slowest. (Overall took about ~2 hours)
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No DNA streaks were visible on the gel. The sample in the first well was the 100bp ladder which did show a substantial streak. I was expecting the supernatants (wells 3-5) to show a little streak if any loose DNA was present and I was not expecting the actual concentrated sample (well 2) to exhibit a streak.
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Tomorrow I will try running a gel with freshly prepared thiol-DNA/ThT/AuNP

Current revision

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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


Image:AliDNAThTAuNPGel.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.

  • Note that for the gel electrophoresis, if using the blue/orange loading dye, allow the gel to run until the lightest blue dye is 3/4 of the way across, this should be the dye that travels the slowest. (Overall took about ~2 hours)

No DNA streaks were visible on the gel. The sample in the first well was the 100bp ladder which did show a substantial streak. I was expecting the supernatants (wells 3-5) to show a little streak if any loose DNA was present and I was not expecting the actual concentrated sample (well 2) to exhibit a streak.

Tomorrow I will try running a gel with freshly prepared thiol-DNA/ThT/AuNP




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